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1.
MINIATURIZED TWO-FOLD DILUTION METHOD FOR ENUMERATING TOTAL MICROBIAL LOAD AND COLIFORMS IN RAW MILK
M. COSTELLO M.S. RHEE D.H. KANG 《Journal of Rapid Methods and Automation in Microbiology》2002,10(1):19-25
A rapid and simple method for enumerating total aerobic plate counts (APC) and coliforms in raw milk was developed and compared with conventional plating method. Following two-fold serial dilution of samples in a 96 well microtiter plate, double strength of two different modified media for APC or coliforms was added to each well. The final positive well (purple to yellow color) was determined and converted to dilution factors. The dilution factor of each sample was converted to Log10 DF (Dilution factors) and compared to actual microbial numbers Log10 CFU/mL. The results of 2-fold dilution method (Log10 DF) were strongly correlated to conventional plating method (Log10 CFU/mL) (P < 0.05). The correlation of the scatterplot of spread plating and 2-fold dilution method indicated a high level of agreement between two methods (R2 = 0.921 for total counts and R2 = 0.916 for forms from raw milk). This 2-fold dilution method is an easy, rapid, and economical method for enumeration of total microbial loads and coliforms in raw milk. 相似文献
2.
Aims: The aim of this study was to develop a sensitive and an effective method suitable for large-scale detection and quantification of Ralstonia solanacearum in soil.
Methods and Results: Based on the specific sequence of R. solanacearum strain G1000, the primer pair R.sol1-R.sol2 and the TaqMan probe Rs-pro were designed, and specific and sensitive PCR detection methods were successfully established. The detection limit was 100 fg μl−1 DNA in conventional PCR and 1·2 fg μl−1 in real-time PCR. By combining real-time PCR with the modified protocols to extract DNA from soil, it was possible to achieve real-time detection of R. solanacearum in soil, and the degree of sensitivity was 100 fg μl−1 . To detect inhibition in soil samples, an exogenous internal positive control (IPC) was included preventing false negative results, and IPC was successfully amplified from all samples tested. The methodology developed was used to detect the presence of R. solanacearum in tobacco fields in China.
Conclusions: The real-time PCR combined with the protocol to extract DNA from soil led to the development of a specific, sensitive and rapid detection method for R. solanacearum in soil.
Significance and Impact of the Study: The real-time PCR improves the detection sensitivity and specificity and provides an important tool for routine detection of R. solanacearum in soil samples and for epidemiological and ecological studies. 相似文献
Methods and Results: Based on the specific sequence of R. solanacearum strain G1000, the primer pair R.sol1-R.sol2 and the TaqMan probe Rs-pro were designed, and specific and sensitive PCR detection methods were successfully established. The detection limit was 100 fg μl
Conclusions: The real-time PCR combined with the protocol to extract DNA from soil led to the development of a specific, sensitive and rapid detection method for R. solanacearum in soil.
Significance and Impact of the Study: The real-time PCR improves the detection sensitivity and specificity and provides an important tool for routine detection of R. solanacearum in soil samples and for epidemiological and ecological studies. 相似文献
3.
Junli Huang Jinzhong Wu Changjun Li Chonggang Xiao Guixue Wang 《Journal of Phytopathology》2010,158(1):15-21
Phytophthora nicotianae is one of the most important soil-borne plant pathogens. A rapid, specific and sensitive real-time polymerase chain reaction (PCR) detection method for P. nicotianae was established, which used primers targeting the internal transcribed spacer (ITS) regions of rDNA genes of Phytophthora spp. Based on the nucleotide sequences of ITS2 of 15 different species of Phytophthora , the primers and probe were designed specifically to amplify DNA from P. nicotianae. With a series of 10-fold DNA dilutions extracted from P. nicotianae pure cultures, the detection limit was 10 pg/μl in conventional PCR, whereas in SYBR Green I PCR the detection limit was 0.12 fg/μl and in TaqMan PCR 1.2 fg/μl, and real-time PCR was 104 –105 times more sensitive than conventional PCR. The simple and rapid procedures maximized the yield and quality of recovered DNA from soil and allowed the processing of many samples in a short time. The direct DNA extractions from soil were utilized to yield DNA suitable for PCR. By combining this protocol with the real-time PCR procedure it has been possible to specifically detect P. nicotianae in soil, and the degree of sensitivity was 1.0 pg/μl. The system was applied to survey soil samples from tobacco field sites in China for the presence of P. nicotianae and the analyses of naturally infested soil showed the reliability of the real-time PCR method. 相似文献
4.
High frequency gene conversion among benomyl resistant transformants in the entomopathogenic fungus Metarhizium anisopliae 总被引:4,自引:0,他引:4
Maurício Reis Bogo Marilene Henning Vainstein francisco JoséLima Aragão Elíbio Rech Augusto Schrank 《FEMS microbiology letters》1996,142(1):123-127
Abstract Three different methods, (i) PEG, (ii) electroporation and (iii) biolistic, were employed to transform Metarhizium anisopliae using benomyl resistance as a selectable marker. Transformation frequencies and mitotic stability varied for each method, from 0.8 to 6.9 transformants μg−1 of DNA and 46%, respectively, by the PEG method; 1.3 to 1.8 transformants μg−1 of DNA and 67% by electroporation; and 32 to 201 transformants μg−1 of DNA and 90% by biolistic. We demonstrate by PCR that 60% of the transformants were generated by gene conversion. 相似文献
5.
Abstract Transport of Mn2+ was repressed in Candida utilis cells grown in continuous culture in high-Mn2+ (100 μM Mn2+ ) medium as compared to cells grown in basic (0.45 μM Mn2+ ) and low-Mn2+ (< 0.05 μM Mn2+ ) media. In contrast, no repression of Cu2+ uptake occurred in high-Cu2+ -grown (25 μM Cu2+ ) cells as compared to cells grown in basic medium (0.54 μM Cu2+ ). Cu2+ -limited cells did not hyperaccumulate Cu2+ and there was not significant difference in initial uptake rates for all 3 Cu2+ conditions. Mn2+ uptake appears to be regulated by a mechanism sensitive to the external Mn2+ concentration, whereas Cu2+ transport is not governed in this way by the external Cu2+ . 相似文献
6.
Abstract: A rapid and effective method is described for electroporation of Brevibacterium sp. R312, a coryneform strain producing nitrile hydratase and amidase. The transformation efficiency of the method is 108 transformants per μg of plasmid under optimal conditions. Parameters optimised included field strength (11.8 kV cm−1 ), pulse length (2.4 ms), plasmid DNA concentration (0.25 μg ml−1 and cell density (1010 cells ml−1 ). Surprisingly, the transformation efficiency did not vary with the growth stage, in contrast to results in the literature. A shuttle vector was constructed containing several unique cloning sites down-stream of the SP6 RNA polymerase promoter. 相似文献
7.
The polymerase chain reaction (PCR) has the potential to detect low levels of the human pathogen Escherichia coli O157 : H7 in bovine faeces. To improve the utility of PCR for this application, several methods for preparing template DNA from bovine faeces, both directly and after non-selective enrichment, were tested. These were boiling, enzyme treatment, enzyme treatment plus phenol-chloroform extraction, and enzyme treatment plus phenol-chloroform extraction plus Geneclean® purification. Of these, the boiling method was the most consistent and had a sensitivity of approximately 3 cfu g−1 faeces, with an assay time of less than 32 h. The boiling method was also combined with immunomagnetic separation (IMS) to detect E. coli O157 : H7 in less than 8 h, but with a sensitivity of approximately 103 cfu g−1 faeces. These methods can be used to prepare template for PCR screening of bovine faeces using any appropriate PCR primers. 相似文献
8.
L.J. Hyman V. Dewasmes I.K. Toth & M.C.M. Pérombelon 《Letters in applied microbiology》1997,25(2):143-147
A simple and sensitive method was developed to replace the need for complex and laborious DNA extraction to remove inhibitory substances in potato tuber peel extract before detection of Erwinia carotovora subsp. atroseptica (Eca) by PCR. Eca was enriched by a factor of 105 when peel extract was inoculated onto a selective medium, CVP, and incubated at 27°C for 24 h. Bacterial micro-colonies which developed were suspended in 500 μl of water and the bacteria diluted in water 100-fold, or 10-fold followed by washing by centrifugation, before PCR testing. The sensitivity of detection obtained with the former was ca 101 –102 cells ml−1 and with the latter ca 101 cells ml−1 , when different numbers of streptomycin-resistant Eca strain were added to peel extract from three Eca-free potato cultivars. The method was validated and the sensitivity confirmed relative to two different commonly used Eca detection methods using naturally contaminated tubers. 相似文献
9.
Renato Fani Marco Bazzicalupo Patrizia Coianiz Mario Polsinelli 《FEMS microbiology letters》1986,35(1):23-27
Abstract Plasmid transformation of the nitrogen-fixing bacterium Azospirillum brasilense is described. A modification of the method of Hanahan [1] was used to transform this bacterium with the 20-kb plasmid pRK290. The efficiency of transformation ranged from 200–1000 transformants per μg of plasmid DNA according to DNA concentration. Ca2+ , Mn2+ and K+ were essential for competence, while Rb+ and hexamine cobalt(III) chloride did not appear necessary. The length and the temperature of heat-pulse during transformation affected the efficiency of transformation. The response to different numbers of plasmid molecules was linear, in the range of 0.05–1.0 μg of DNA. No transformants were obtained with pRK290 plasmid DNA linearized with Eco RI. The transformability of different strains of Azospirillum has been compared. 相似文献
10.
Several nuclear events seen during the cleavage period in Eudorina suggest that chromosome endoreduplication, proportional to the number of cells to be produced, may occur in the gonidia prior to cleavage. Presumably the DNA concentration is reduced to the haploid level during rapid, successive divisions of the cleavage period.
To test this hypothesis, I determined DNA content of gonidia as they grew from 4 μm to 38 μm in diameter between cleavage periods. During growth from 4 μm to 8 μm in diameter, the DNA concentration remained at the haploid level of 0.17 pg/cell. As gonidia in 64 cell colonies continued to grow from 8 μm to 33 μm in diameter, their DNA concentrations increased 60-fold.
Analysis of the Eudorina DNA by equilibrium centrifugation in CsCl showed only 2 bands with buoyant densities of 1.721 g/cm3 and 1.699 g/cm3 , presumed to be nuclear and chloroplast, respectively, on the basis of labelling with 3 H-thymidine and 3 H-adenine. The 8: 2 ratio of the two bands did not change with increase in cell size and no other bands were detected, suggesting that both nuclear and chloroplast DNAs were synthesised proportionately prior to the cleavage period. 相似文献
To test this hypothesis, I determined DNA content of gonidia as they grew from 4 μm to 38 μm in diameter between cleavage periods. During growth from 4 μm to 8 μm in diameter, the DNA concentration remained at the haploid level of 0.17 pg/cell. As gonidia in 64 cell colonies continued to grow from 8 μm to 33 μm in diameter, their DNA concentrations increased 60-fold.
Analysis of the Eudorina DNA by equilibrium centrifugation in CsCl showed only 2 bands with buoyant densities of 1.721 g/cm
11.
The first highly efficient protocol is described for the electrotransfection of Propionibacterium freudenreichii with DNA phage. The transfection efficiency is 7 times 105 transfectants per μg of DNA under optimal conditions. Optimized parameters included the field strength (12.5 kV, 200 Ohms, 25 μF), phage DNA concentration (1 μg ml-1 ) and cell density (1.5 times 1010 cells ml-1 ). Growth in the presence of glycine and harvesting of cells during the early exponential growth phase increased the transfection efficiency. This electrotransfection protocol is of importance for the genetic improvement of dairy propionibacteria. 相似文献
12.
Abstract— Ouabain (200μ m ) inhibited incorporation of radiolabelled leucine or glycine into the protein of neonatal synaptosome fractions but had minimal effect on preparations from adult rats. Leucine uptake into synaptosomes was rapid but not influenced by 200μ m -ouabain in contrast to ouabain inhibition of [14 C]glycine and [14 C]γ-aminobutyric acid uptake. Ouabain blocked the Na+ -dependent (stimulated) component of synaptosome fraction protein synthesis in the presence of 25m m -K+ . Ouabain inhibition was not alleviated by addition of ADP or ATP. 100μ m -atractylate failed to influence [3 H]leucine uptake or incorporation. Synergistic inhibition by ouabain was observed with the cycloheximide-sensitive component of protein synthesis and the chloramphenicol sensitive phase. Increasing the medium Ca2+ concentration stimulated protein synthesis and this stimulated component was inhibited by ouabain. Ouabain inhibition was associated with decreasing intraterminal K+ concentration and [K]i was linearly related to the protein synthesis rate in control and ouabain treated preparations. 相似文献
13.
Abstract An optimized polyethylene glycol (PEG) method of transformation was developed for Methanococcus maripaludis using the pKAS102 integration vector. The frequency of transformation with 0.8 μg of plasmid and 3×109 cells was 4.8×10−5 transformants cfu−1 , or 1.8×105 transformants μg−1 , which was four orders of magnitude greater than with the natural transformation method. A Pst I restriction activity in M. maripaludis was also identified. Methylation of the plasmid with Pst I methylase increased the methanococcal transformation frequency at least four-fold. Also, chromosomal DNA from M. maripaludis was resistant to digestion by the Pst I endonuclease. 相似文献
14.
Transformation of group A streptococci by electroporation 总被引:1,自引:0,他引:1
Abstract The introduction, via electroporation, of free plasmid DNA into three strains of Streptococcus pyogenes is described. The method is very simple and rapid and efficiencies vary from 1 × 103 to 4 × 104 per μg of DNA. The method was also used to introduce an integrative plasmid and transformants were obtained, albeit at a somewhat lower frequency (2 × 102 ). Some of the plasmids used in this study are derivatives of the Lactococcus lactis subsp. cremoris Wg2 plasmid pWV01. These broad host range vectors replicate in Gram-positives as well as Gram-negatives (viz. Escherichia coli ). Here we show that they also replicate in S. pyogenes and S. sanguis . 相似文献
15.
An in vitro method of growing bacteria as a defined nutrient-depleted biofilm is proposed. The medium was defined nutritionally in terms of the quantitative composition and by the total amount of nutrient required to achieve a defined population size. Escherichia coli and Burkholderia cepacia were incubated on a filter support placed on a defined volume of solid medium. The change of biomass of the biofilm population was compared with the change in a planktonic culture. The size of the population in stationary phase was proportional to the concentration of limiting substrate up to 40 μmol cm−2 glucose for E. coli and up to 2·7 × 10−9 mol cm−2 iron for B. cepacia . Escherichia coli growing exponentially had a growth rate of μ = 0·30 h−1 in a biofilm and μ = 0·96 h−1 in planktonic culture. The growth rate, μ, for exponentially growing B. cepacia in a biofilm was 1·12 h−1 and in planktonic culture 0·78 h−1 . This method allows the limitation of the size of a biofilm population to a chosen value. 相似文献
16.
Nickel and rubidium uptake by oat plants ( Avena sativa L. cv. Victory) were examined in relation to solution temperature, solution concentrations, metabolic inhibitors, anaerobic root conditions, transpiration and time. Over a 4-h period, uptake rates for both Ni2+ and Rb+ remained constant at 23°C. Decreasing temperatures to 2°C, 20 μ M concentrations of 2,4-dinitrophenol (DNP), or anaerobic root conditions decreased Ni2+ and Rb+ uptake rates by 97 to 86% in whole plants. Treatment of excised roots with 20 μ M DNP decreased Ni2+ uptake by 93%. Nickel and Rb+ uptake rates measured as a function of the external solution concentration followed a typical parabolic curve. Km (0.012 m M ) and Vmax [2.72 μmol (g dry weight)-1 h-1 ] values for Ni2+ were nearly 7 times lower than those for Rb+ [0.09 m M and 19.2 μmol (g dry weight)-1 h-1 ]. In all experiments, Ni2+ and Rb+ showed qualitatively similar uptake patterns, but Rb+ uptake was quantitatively more sensitive than Ni2+ to experimental manipulations. 相似文献
17.
Determination of the surface area of fine tomato roots via cation uptake experiments 总被引:1,自引:0,他引:1
H. T. Wolterbeek 《Physiologia plantarum》1987,69(4):704-708
A method is described by which the surface area of a root is estimated from cation uptake data. 24 Na+ was supplied to excised roots of tomato ( Lycopersicon esculentum Mill, cv. Tiny Tim) seedlings at 3 μ M in unstirred solution. Coarse roots, for which external surface area and specific gravity could be measured accurately, were used to estimate the thickness (dN ) of the Nernst boundary layer at the root surface. 24 Na+ uptake (J1 ) was measured by γ-ray spectroscopy. Jt and dN were used to calculate the total surface area for ion absorption in fine roots, assuming that Na uptake rate was diffusion-limited. The results were compared to data obtained by conventional methods and indicated the usefulness of the cation uptake technique for quantitative estimates of root surfaces. 相似文献
18.
A rapid and sensitive ion chromatographic technique for the determination of sulfate and sulfate reduction rates in freshwater lake sediments 总被引:6,自引:0,他引:6
Abstract Newly developed low capacity columns were used in suppressed ion chromatography for rapid and highly reproducible determination of SO4 2− in porewater samples from freshwater sediments without preconcentration of samples. With a 50 μl injection the detection limit for SO4 2− was ca. 50 pmol (= 1 μ M) with a precision of 1–3% at the 10–200 μM level and <1% at concentrations above 200 μM. SO4 2− could be measured in 4–5 min with the routinely used eluent (3.0 mM NaHCO3 /0.8 mM Na2 CO3 ). When the strength of the eluent was increased to 3.0 mM NaHCO3 /2.0 mM Na2 CO3 , sulfate analysis was possible in less than 3 min, provided that samples were nitrate-free. Under these conditions S2 O3 2− could also be sensitively determined in about 6 min. Examples of application of the method are given for measurements of sulfate reduction rates in freshwater sediment samples from Lake Constance. 相似文献
19.
Anne Ojala 《Nordic Journal of Botany》1993,13(6):697-705
The effects of light and temperature on cell size and cellular composition (chlorophyll, protein, carbohydrate) of two freshwater cryptophytes were studied with batch cultures. Neither of the species had a constant cell size but the size varied with growth conditions. At each temperature the smallest cells were recorded at the lowest experimental photon flux density. The smallest cells of Cryptomonas 979/67 had an average volume of 232 μm3 and the largest ones 1 020 μm3 . In Cryptomonas 979/62 the smallest and largest cells measured 4 306 μm3 and 12 450 μm3 . Both species increased their cellular chlorophyll content when PFB dropped below 110–120 μmol m-2 s-1 . The highest and lowest chlorophyll contents of 979/67 were 7.45 fg μm-3 and 0.55 fg μm-2 respectively. For 979/62 the corresponding values were 10.23 fg μm-3 and 0.93 fg μm-3 . In both species the protein content remained stable at PFDs higher than 110–120 μmol m-2 S-1 . The highest content of protein measured in 979/67 was 638 fg μm-3 and the lowest 147 fg μm-3 . For 979/62 these values were 1 036 fg μm-3 and 148 fg μm-3 respectively. The carbohydrate results were less clear and no pattern either in response to photon flux density or temperature was obvious. The lowest and highest contents recorded for 979/67 were 62 fg μm-3 and 409 fg μ-3 and for 979162, 36 fg μm-3 and 329 fg μm-3 相似文献
20.
C.A. FROELICH I.B. ZABALA DÍAZ S.C. RICKE 《Journal of Rapid Methods and Automation in Microbiology》2002,10(3):161-172
A potential rapid bioassay for methionine hydroxy analog (MHA) feed additive (ALIMET®) was examined using a methionine auxotroph E. coli strain. Bacterial cells were grown in minimal media containing a concentration range of 0 to 26.8 μM of either L-methionine or MHA as ALIMET®. Increasing either methionine or MHA concentration increased the growth rate of the methionine auxotroph. The estimated substrate affinities for methionine compared to MHA were not significantly different (P > 0.13) and the maximum growth rate estimates were also similar (P > 0.34). Methionine and MHA standard curves yielded linear responses (R2 = 0.96) to increasing concentrations of the respective substrate. Based on these results it appears that the E. coli methionine auxotroph would have potential utility for further development of a rapid bioassay of ALIMET®. 相似文献