Bead Mill Homogenization Research Articles

Erratum to “The properties of cement paste incorporating multi wall carbon nanotube solution prepared through bead mill and high-pressure homogenizer process with polycarboxylate ester” [J. Constr. Build. Mater. 337 (2022) 127598

Erratum to “The properties of cement paste incorporating multi wall carbon nanotube solution prepared through bead mill and high-pressure homogenizer process with polycarboxylate ester” [J. Constr. Build. Mater. 337 (2022) 127598

The Properties of Cement Paste Incorporating Multi Wall Carbon Nanotube Solution Prepared through Bead Mill and High-Pressure Homogenizer Process with Polycarboxylate Ester

Carbon nanotube (CNT) has been successfully used for production of cementitious composites due to its excellent mechanical properties. In most of the existing literatures, the dispersion of CNT was mostly achieved using either ultrasonic water bath or tip sonicator that is known to provide relatively small amount of energy. In this work, the high energy dispersion processes, bead mill and high-pressure homogenizer, which have been applied for other industrial purposes, were applied to prepare multi wall carbon nanotube (MWCNT) solution with polycarboxylate ester (PCE) as a dispersing agent. The MWCNT solution was used for preparation of cement paste specimens. Ultrasonic tip sonication, a conventional lab-scale dispersion process, was also used for comparison purposes. According to the experimental results, PCE in MWCNT solution behaved as an air-entraining high-range water reducer for cement paste, causing increase in porosity, retardation in hydration, and reduction in undisturbed yield stress. Such effect was more significantly observed from the cases with bead mill and high-pressure homogenizer than ultrasonic tip sonication. The 28-day compressive strength increased by the addition of MWCNT, showing the maximum efficiency at 0.05% MWCNT content. The viscosity of cement paste increased as the amount of MWCNT increased, but flexural strength generally decreased as MWCNT was incorporated.

Impact of Different Mixing Methods on the Performance of Suspension-Based Transdermal Delivery Systems.

Suspension-based matrix transdermal delivery systems (TDSs) are specialized systems that maintain a continuous driving force for drug delivery over prolonged wear. The pressure-sensitive adhesive (PSA) is the most critical constituent of such systems. Our study aimed to determine the effect of different mixing methods on the performance of silicone PSA-based suspension TDSs. Lidocaine suspension TDSs were prepared using conventional slow rotary mixing, high-speed homogenization, bead-mill homogenization, vortex shaking, and by an unguator. Resultant TDSs were tested for tack, shear, and peel properties and correlated to coat weight, content uniformity, microstructure, and in vitro permeation across dermatomed human skin. Every mixing method tested caused a significant reduction in peel. However, bead-mill homogenization resulted in significant loss of all adhesive properties tested, while unguator-mixed TDSs retained most properties. Good linear correlation (R2 = 1.000) between the shear properties of the TDSs with the average cumulative amount of lidocaine permeated after 24 h was observed, with no significant difference between percutaneous delivery from slow rotary-mixed systems (1334 ± 59.21 μg/cm2) and unguator-mixed systems (1147 ± 108.3 μg/cm2). However, significantly lower delivery from bead-mill homogenized systems (821.1 ± 28.00 μg/cm2) was noted. While many factors affect TDS performance, careful consideration must also be given to the processing parameters during development as they have been shown to affect the resultant system's therapeutic efficacy. Extensive mixing with bead-mill homogenization demonstrated crystallization of drug, loss in adhesive properties, coat weight, and film thickness, with reduced transdermal delivery of lidocaine from the prepared system.

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans.

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitateC. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

Impact of Sampling and Storage Techniques on Beef Muscle Measurements During Aging

ObjectivesThis objective was to determine if variations in beef sampling techniques utilized by meat researchers have a significant impact on beef muscle measurements during aging.Materials and MethodsTen beef short loins (IMPS 180) were purchased from a commercial packing plant within 48 h of slaughter. Loins were transported to the NDSU Meat Science laboratory where they were mapped into four sections from most anterior (1) to most posterior (4). Within sections, two, 40-g samples were removed; one sample was vacuum packaged (SMALL-VAC) and the other sample was stored in a wire-closure sealed bag (SMALL-BAG). The remaining whole short loin was vacuum packaged. All samples and whole short loins were stored at 4°C for 10 d. At 10 d, the short loins were sampled again where one, 40-g sample was removed from each mapped section (WHOLE-VAC). Purge loss was measured by weighing each sample prior to packaging treatment and at the end of the 10-d aging period; percentage change in weight was calculated. Troponin-T degradation was determined by western blot. Briefly, protein was extracted in an SDS-phosphate buffer, separated by SDS-PAGE under reducing conditions, and transferred to PVDF membranes. Western analysis was done using an anti-troponin-T antibody (clone JLT 12), and immunoreactive bands (Band 1 = doublet ∼42 to 45 kDa; Band 2 = doublet ∼ 36 to 38 kDa, Band 3 = 30 kDa) were analyzed for differences in density. Sarcomere length was determined using HeNe laser diffraction. Thinly sliced samples (∼50 to 100 mg) were placed in a sucrose-phosphate buffer and subjected to beadmill homogenization. A drop of the homogenate was placed on a glass slide, diffraction patterns were measured, and sarcomere length was calculated. Thiobarbituric acid reactive substances (TBARS) were assessed using a colorimetric assay. Analysis was conducted using Proc Mixed procedure of SAS where storage type, section location, and their interaction were used as fixed effects.ResultsThere was a storage type by section interaction (P = 0.017) that occurred with purge loss. SMALL-VAC samples released more purge than SMALL-BAG from the more posterior samples. Troponin-T Band 1 tended to be less (P = 0.07) in WHOLE-VAC samples compared with SMALL-VAC and SMALL-BAG. There was a storage type by section interaction (P = 0.02) where the most posterior SMALL-BAG samples had greater Band 2. There were no differences (P ≥ 0.25) in Band 3 between treatments. There was no difference (P = 0.29) in sarcomere length due storage type. However, there was a difference (P = 0.01) in sarcomere length between sections, where the shortest sarcomeres were in the center of the strip loin and longest sarcomeres on either end. There was a storage type by section interaction (P = 0.02) for TBARS where concentration was greatest in the most posterior portion of SMALL-BAG compared with WHOLE-VAC.ConclusionCollection of smaller samples for aging studies may not be representative of samples aged in a whole primal cut and may influence research outcomes.

A Method for the Activation of Platelet-Rich Plasma via Bead Mill Homogenizer for Mesenchymal Stem Cell Culture.

Activated platelet-rich plasma (PRP) has been studied as a replacement for fetal bovine serum (FBS) in stem cell culture. However, current methods are time-consuming or require addition of exogenous substances to activate PRP, which have disadvantages in clinical applications. In this study, we developed a new method for PRP activation using a bead mill homogenizer and compared it with previous methods of PRP activation. PRP was prepared via a two-step centrifugation process and activated via calcium (Ca-PRP), freeze-thaw cycles (FT-PRP), or bead mill homogenizer processing (BM-PRP). Quantification of growth factors in PRP revealed that all forms of activated PRP released higher levels of platelet-derived growth factor-AB and transforming growth factor-β1 than those in platelet-poor plasma; however, BM-PRP resulted in significantly higher levels of growth factors than those from Ca-PRP and FT-PRP. Next, we analyzed the ability of the various forms of PRP to stimulate proliferation, migration, and differentiation of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). Our results showed that BM-PRP significantly increased proliferation and migration rates of UCB-MSCs while maintaining the phenotypical properties and stem cell abilities of MSCs. Therefore, the developed method could be suitable for PRP activation, and the BM-activated PRP could be an adequate replacement for FBS in stem cell culture.

Enhancement of the in vitro penetration of quercetin through pig skin by combined microneedles and lipid microparticles

Silicon microneedle patches were investigated, alone or in combination with lipid microparticles (LMs), as a system to improve the in vitro skin penetration of the antioxidant flavonoid, quercetin. LMs loaded with quercetin were prepared by melt emulsification and sonication. The flavonoid content of LMs was 11.7±0.3% and their mean diameter and polydispersity index were 8.1μm and 0.66, respectively. Emulsions containing quercetin, free or microencapsulated, were applied to untreated- or microneedle-treated pig skin mounted in Franz diffusion cells. The amount of flavonoid penetrated into the stratum corneum and viable epidermis were measured by HPLC, after validated tape-stripping and bead mill homogenization procedures, respectively. Compared to intact skin, a marked increase in quercetin levels permeated into the stratum corneum (from 1.19±0.12μg/cm2 to 2.23±0.54μg/cm2) and viable epidermis (from 0.10±0.01μg/cm2 to 0.56±0.27μg/cm2) was achieved when skin was treated with the flavonoid-loaded LMs in combination with microneedle arrays. Conversely, perforation of the cutaneous surface by microneedles did not produce any significant improvement in the skin penetration of non-encapsulated quercetin. The enhanced (5.5-fold) intra-epidermal delivery of quercetin attained by the LM/microneedle strategy described here, is particularly relevant since the main quercetin site of action is in the epidermis.

Evaluation of mRNA extraction methods from murine skin (776.4)

Intro: Transcriptome research has become increasingly popular to monitor global changes in gene expression. In particular, transcriptome differences between diseases versus healthy states can provide valuable insight into understanding immune response mechanisms. However, the isolation of high‐quality mRNA from tough tissues, such as skin, can represent a significant challenge in research. Herein, we compare two methods for mRNA extraction from two murine skin types with a specific focus on mRNA yields and integrity.Methods: 1cm2 full‐thickness mouse skin (epidermal and dermal layers) samples were freshly harvested, separated from fat, and kept on ice until homogenization. Samples were cut into fragments and placed in 1ml Trizol, then homogenized using a manual Dounce homogenizer on ice, or an automated Omni Bead Ruptor 24 (BR24) bead mill homogenizer without refrigeration. Clarified sample supernatants were mixed with EtOH to precipitate nucleic acids, and sample yields and quality were determined by spectrophotometry. The mRNA purity and integrity of each sample was additionally determined using an Agilent BioAnalyzer.Results: Spectrophotometric data indicated that, depending on skin tissue type and sample size, mRNA yield from samples processed by BR24 versus Dounce was increased 2‐6 fold. The 260/280 ratios were equivalent across both methods; however, the Alginet electropherograms revealed that Dounce homogenization produced samples with unacceptably high MW contaminant DNA and low MW degradation species, while the BR24 processing produced superior mRNA samples of outstanding purity and RNA integrity (RIN). (Omni BR24 RIN values 8.4 ‐ 8.8 ; Dounce RIN, 蠄 4.3). Thus, high quality mRNA can be efficiently isolated from difficult to disrupt tissues such as skin.

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

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NovelChlamydia-like Organisms as Cause of Bovine Abortions, UK

Novel<i>Chlamydia</i>-like Organisms as Cause of Bovine Abortions, UK

Comparison of dry- and wet-based fine bead homogenizations to extract DNA from fungal spores

The present study explored DNA extraction kinetics from fungal spores, i.e., Aspergillus niger, Penicillium chrysogenum and Cladosporium sphaerospermum, by fine bead mill homogenization. In particular, the study aimed to investigate basic differences between the dry- and wet-based methods. The results showed higher initial rates of the DNA extractions by the dry-based method than by the wet-based method, due to higher collision efficiency among fine beads and fungal spores. Based on the experimental results, we constructed kinetic models. While the results by the wet-based method were fitted well with an existing first-order release-degradation model, the results by the dry-based method were not fitted well. Meanwhile, a newly constructed first-order release-degradation model, assuming a proportion of the DNA remained inside the disrupted spore cells and protected from further sheer stress, showed good correlations. The real-time PCR assays showed the PCR efficiencies of the DNA obtained by the dry-based method were higher than those by the wet-based method likely due to increased moderate fragmentation of the DNA by the dry-based method. Thus, although wet-based methods have been commonly used, dry-based methods might also be applicable to achieve efficient extraction and PCR amplification of fungal DNA.

Tribological Behavior of Lubricating Oil-Based NanofluidsContaining Ag and Carbon Nanoparticles

Oil-based nanofluids were prepared by dispersing Ag, graphite and carbon black nanoparticles in lubricating oil. Agglomerated nanoparticles were dispersed evenly with a high-speed bead mill and/or ultrasonic homogenizer, and the surfaces of the nanoparticles were modified simultaneously with several dispersants. Their tribological behaviors were evaluated with a pin-on-disk, disk-on-disk and four-ball EP and wear tester. It is obvious that the optimal combination of nanoparticles, surfactants and surface modification process is very important for the dispersity of nanofluids, and it eventually affects the tribological properties as a controlling factor. Results indicate that a relatively larger size and higher concentration of nanoparticles lead to better load-carrying capacity. In contrast, the use of a smaller size and lower concentration of particles is recommended for reducing the friction coefficient of lubricating oil. Moreover, nanofluids with mixed nanoparticles of Ag and graphite are more suitable for the improvement of load-carrying capacity and antiwear properties.

Quantitative detection of Beauveria bassiana GHA (Ascomycota: Hypocreales), a potential microbial control agent of the emerald ash borer, by use of real-time PCR

Accurate monitoring of an introduced, mass-released microbial control agent is essential in evaluating its persistence and in designing application strategies for insect pest control. As part of our multi-year study on the development and use of the entomopathogenic fungus Beauveria bassiana against the emerald ash borer, a major invasive pest of ash trees, we are determining persistence of the fungus sprayed on ash trees and leached onto soil. We developed a real-time PCR assay to detect and quantify B. bassiana strain GHA, the active ingredient in the mycoinsecticide utilized in our field studies. Real-time PCR primers and probe, based on a 445-bp sequence characterized amplified region fragment unique to GHA, generated a 96-bp fragment that was also specific to this strain. We also developed a DNA extraction method to maximize accuracy of quantification from environmental samples. DNA from fungal conidia in soil samples or recovered from leaf and bark washings was extracted using bead mill homogenization followed by purification of the crude extract using Sephadex-polyvinylpolypyrrolidone microcolumns. Sensitivity assays comparing quantity of GHA DNA detected between pure and mixed samples showed that background genomic DNA from bark and leaf did not significantly affect detection sensitivity. However, background soil DNA and likely remaining co-extracted contaminants reduced sensitivity of assays at 400pg or less of GHA DNA. Comparison of various conidial titer on ash bark and leaves versus predicted values based on real-time PCR showed that estimates were best obtained from samples with high titer of the fungus.

Effect of Preparation Conditions on Thermal and Electrical Properties of Oil-Based Nanofluids for Transformer Application

Oil-based nanofluids were prepared by dispersing nonconducting fibrous <TEX>$Al_2O_3$</TEX> and spherical AlN nanoparticles in transformer oil. In this study, the effects of wet grinding and surface modification of particles on thermal and electrical properties of nanofluids were investigated. Grinding experiments were conducted with high-speed bead mill and ultrasonic homogenizer and nanoparticles were surface modified by oleic acid and polyoxyethylene alkyl acid ester(PAAE) in n-hexane or transformer oil, at the same time. It is obvious that the combination of nanoparticle, dispersant and dispersion solvent is very important for the dispersity of nanofluids. For nanofluids containing 1.0vol.% AlN particles in transformer oil, the enhancement of thermal conductivity was 11.6% compared with pure transformer oil. However, the electric-insulating property of AlN nanofluids was very low due to used dispersant itself. Therefore, the effect of the dispersant on thermal/electrical/physical properties of the transformer oil should be considered before selecting a proper dispersant.

Detection and quantification of Entomophaga maimaiga resting spores in forest soil using real-time PCR

Environmental sampling to monitor entomopathogen titre in forest soil, a known reservoir of insect pathogens such as fungi and viruses, is important in the evaluation of conditions that could trigger epizootics and in the development of strategies for insect pest management. Molecular or PCR-based analysis of environmental samples provides a sensitive method for strain- or species-based detection, and real-time PCR, in particular, allows quantification of the organism of interest. In this study we developed a DNA extraction method and a real-time PCR assay for detection and quantification of Entomophaga maimaiga ( Zygomycetes: Entomophthorales), a fungal pathogen of the gypsy moth, in the organic layer of forest soil. DNA from fungal resting spores (azygospores) in soil was extracted using a detergent and bead mill homogenization treatment followed by purification of the crude DNA extract using Sephadex–polyvinylpolypyrrolidone microcolumns. The purification step eliminated most of the environmental contaminants commonly co-extracted with genomic DNA from soil samples but detection assays still required the addition of bovine serum albumin to relieve PCR inhibition. The real-time PCR assay used primers and probe based on sequence analysis of the nuclear ribosomal ITS region of several E. maimaiga and two E. aulicae strains. Comparison of threshold cycle values from different soil samples spiked with E. maimaiga DNA showed that soil background DNA and remaining co-extracted contaminants are critical factors determining detection sensitivity. Based on our results from comparisons of resting spore titres among different forest soils, estimates were best for organic soils with comparatively high densities of resting spores.

A Comparison of Cell Wall Disruption Techniques for the Isolation of Intracellular Metabolites from Pleurotus and Lepista sp.

Different techniques were compared for their effectiveness in the disruption of the rigid cell walls of Basidiomycetes. Grinding under liquid nitrogen, stirred glass bead milling and enzymatic cell lysis were applied to the mycelia of Pleurotus sapidus and Lepista irina grown submerged. Each of the disruption procedures was evaluated by testing the quantity and quality of released intracellular metabolites: DNA, RNA, enzymes, and secondary metabolites. The most suitable method for nucleic acid isolation was grinding under liquid nitrogen, while bead mill homogenization was the superior technique for isolation of active enzymes. A new effective method is proposed for isolation of secondary metabolites with the aid of bead milling of fungal mycelia.

A molecular approach to the characterization of the eukaryotic communities of an extreme acidic environment: Methods for DNA extraction and denaturing gradient gel electrophoresis analysis

The diversity of the phytobenthonic community present in six acidophilic microbial mats from Río Tinto (Iberian Pyritic Belt, SW Spain) was analysed by optical microscopy and two molecular techniques, denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 18S rDNA cloned gene fragments. Sixteen DNA isolation protocols as well as two commercial DNA extraction kits were tested and their efficiency compared. Purified DNA extracts were amplified by PCR using universal eukaryotic primers and the PCR products analysed by DGGE. Bead-mill homogenization was found to be superior to the other cell lysis methodologies assayed (sonication or freeze-thawing cycles) as it allowed efficiencies of cell disruption of over 95%. The methods combining bead-mill homogenization in the presence of SDS, treatment with chemical extractants (hexadecylmethylammonium bromide or guanidine isothiocyanate) and phenol extraction resulted in DNA preparations that amplified the same number of bands when analysed by DGGE as the two commercial kits assayed. The phylogenetic affiliations of the DGGE bands were determined by a BLAST search, and nine different species related to the Chlorophyta, Ciliophora, Kinetoplastida, Ascomycota, Streptophyta and Colcochaetales taxonomical groups were identified. Similar levels of diversity were found using cloning procedures. Although not all the species observed under the microscope were detected using molecular techniques, e.g. euglenas, heliozoan, or amoebae, DGGE fingerprints showed rather well the level of diversity present in the samples analysed, with limitations similar to cloning techniques.

Quantitative Measurement of Fungal DNA Extracted by Three Different Methods Using Real-Time Polymerase Chain Reaction

Quantification of fungal populations in the environment is important for gaining a better understanding of various microbial processes. Recently, the development of real-time quantitative PCR (RTQ-PCR) has eliminated the variability associated with conventional quantitative PCR, thereby allowing the routine and reliable quantification of PCR products. Thus, in this present study, RTQ-PCR was used to quantify the fungal target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in the presence of liquid nitrogen, and hot detergent SDS based enzymatic lysis combined with bead beating) to determine the suitability of the quantification of target DNA. For the purpose of this study, pure culture of Aspergillus fumigatus (model organism), sterilized soil seeded with a known amount of A. fumigatus (model soil system), and woodland and grassland soil samples (environmental samples) were chosen to extract DNA by the above three different protocols. The extracted DNA was then quantified by spectroscopy and a RTQ-PCR system. 18S rDNA specific universal fungal primers were used to quantify the target part and then amplification products were verified by agarose gel electrophoresis. Standard curves used for the quantification by RTQ-PCR revealed strong linear relationships ( R 2 =0.9994 for the primer pair NS1 and NS2 and 0.9938 for the primer pair nu-SSU-0817 and nu-SSU-1196) with a higher amplification efficiency, e =0.983 for the primer pair NS1 and NS2 and 0.956 for the primer pair nu-SSU-0817 and nu-SSU-1196. Although for pure culture the hot detergent SDS based enzymatic lysis combined with bead beating method showed the highest target DNA copy number (1.5 × 10 9 copies/μl), for the model soil system and both environmental samples the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (6.16 × 10 8 and 2.7 × 10 8 copies/μl for woodland and grassland, respectively), high DNA yield (6.4 μg/g and 1.8 μg/g of soil for woodland and grassland, respectively), and high recovery on the basis of the target DNA copy number (39.2%), suggesting an overall high extraction efficiency.

Quantitative measurement of fungal DNA extracted by three different methods using real-time polymerase chain reaction

Quantification of fungal populations in the environment is important for gaining a better understanding of various microbial processes. Recently, the development of real-time quantitative PCR (RTQ-PCR) has eliminated the variability associated with conventional quantitative PCR, thereby allowing the routine and reliable quantification of PCR products. Thus, in this present study, RTQ-PCR was used to quantify the fungal target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in the presence of liquid nitrogen, and hot detergent SDS based enzymatic lysis combined with bead beating) to determine the suitability of the quantification of target DNA. For the purpose of this study, pure culture of Aspergillus fumigatus (model organism), sterilized soil seeded with a known amount of A. fumigatus (model soil system), and woodland and grassland soil samples (environmental samples) were chosen to extract DNA by the above three different protocols. The extracted DNA was then quantified by spectroscopy and a RTQ-PCR system. 18S rDNA specific universal fungal primers were used to quantify the target part and then amplification products were verified by agarose gel electrophoresis. Standard curves used for the quantification by RTQ-PCR revealed strong linear relationships ( R 2=0.9994 for the primer pair NS1 and NS2 and 0.9938 for the primer pair nu-SSU-0817 and nu-SSU-1196) with a higher amplification efficiency, e=0.983 for the primer pair NS1 and NS2 and 0.956 for the primer pair nu-SSU-0817 and nu-SSU-1196. Although for pure culture the hot detergent SDS based enzymatic lysis combined with bead beating method showed the highest target DNA copy number (1.5 × 10 9 copies/μl), for the model soil system and both environmental samples the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (6.16 × 10 8 and 2.7 × 10 8 copies/μl for woodland and grassland, respectively), high DNA yield (6.4 μg/g and 1.8 μg/g of soil for woodland and grassland, respectively), and high recovery on the basis of the target DNA copy number (39.2%), suggesting an overall high extraction efficiency.