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Mycoplasma contamination of cell cultures is a common problem that can affect the growth, metabolism, and function of cultured animal cells. The ability to detect mycoplasma in cell cultures provides an opportunity to ensure that cells are free of contamination, and to replace those that are not. For additional information, see Practices E 1531, E 1532, and E 1536. Strict adherence to established, well-tested procedures is necessary. This practice was developed by Task Group E48.01.02 to assist in developing and maintaining an established regimen for mycoplasma detection by indirect 4′-6-Diamidino-2-Phenylindole (DAPI) fluorochrome staining.This practice is intended for use in examining cultured animal cells for the presence of mycoplasma contamination.This practice is not intended for use in the detection of mycoplasma contamination in serum, culture media, or systems other than cultures of animal cells.All cell cultures to be examined for mycoplasma should undergo a minimum of two passages in antibiotic-free tissue culture medium before testing.1.1 This practice covers procedures used for the detection of mycoplasma contamination by indirect DNA staining.1.2 This practice does not cover direct methods for the detection of mycoplasma or other indirect methods such as enzymatical detection or DNA probes.1.3 This practice does not cover methods for the identification of mycoplasma organisms.1.4 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

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4.1 The Manual Observer-Dependent Assay—The manual quantification of cell and CFU cultures based on observer-dependent criteria or judgment is an extremely tedious and time-consuming task and is significantly impacted by user bias. In order to maintain consistency in data acquisition, pharmacological and drug discovery and development studies utilizing cell- and colony-based assays often require that a single observer count cells and colonies in hundreds, and potentially thousands of cultures. Due to observer fatigue, both accuracy and reproducibility of quantification suffer severely (5). When multiple observers are employed, observer fatigue is reduced, but the accuracy and reproducibility of cell and colony enumeration is still significantly compromised due to observer bias and significant intra- and inter-observer variability (2, 4) . Use of quantitative automated image analysis provides data for both the number of colonies as well as the number of cells in each colony. These data can also be used to calculate mean cells per colony. Traditional methods for quantification of colonies by hand-counting coupled with an assay for cell number (for example, DNA or mitochondrial) remains a viable method that can be used to calculate the mean number of cells per colony. These traditional methods have the advantage that they are currently less labor intensive and less technically demanding (8, 9). However, the traditional assays do not, provide colony level information (for example, variation and skew), nor do they provide a means for excluding cells that are not part of a colony from the calculation of mean colony size. As a result, the measurement of the mean number of cells per colony that is obtained from these alternative methods may differ when substantial numbers of cells in a sample are not associated with colony formation. By employing state-of-the-art image acquisition, processing and analysis hardware and software, an accurate, precise, robust and automated analysis system is realized.4.1.1 Areas of Application—Cell and colony enumeration (CFU assay) is becoming particularly important in the manufacture, quality assurance/control (QA/QC), and development of product safety and potency release criteria for cell-based regenerative medicine and cellular therapy. The U.S. Food and Drug Administration (FDA) has a guidance document that indicates that the CFU assay may be appropriate for testing stability of placental and umbilical cord blood-derived stem cells (7). Since cell source validation and QA/QC comprise approximately 50 % of the manufacturing cost of cellular therapies (10), developing a precise, robust, and cost-effective means for enumerating cells and colonies is vital to sustainability and growth in this industry. The broad areas of use for automated analysis of colony forming unit assays include:4.1.1.1 Characterization of a cell source by correlating biological potential and functional potency with CFU formation.4.1.1.2 Characterization of the effect of processing steps or biological or physical manipulation (for example, stimuli) on cells or colony formation.4.1.1.3 Cell and colony characterization using specific fluorescent and non-fluorescent (differentiation) markers.4.1.1.4 Extrapolation of the biological potency (for example, differentiation, proliferative, and so forth) of a larger sample from application of colony forming assay to sub-samples.4.1.1.5 Provision of criteria for sub-colony selection of preferred colonies (specific tissue type, proliferation rate, and so forth) for use and/or further expansion.4.2 The Technology (image acquisition, processing, and analysis)—Current standards utilize user input for defining the presence and location of colonies based on visualization of an entire culture surface at low magnification through the eyepieces of a microscope. In this case, the sample may be viewed in transmission light mode (unstained or with a histochemical marker) or fluorescently with a dye or antibody. For this practice, the colony count is the only measurable output parameter. Utilizing a microscope-based imaging system to stitch together high resolution image tiles into a single mosaic image of the entire culture surface and subsequently “clustering” segmented cells using image processing algorithms to delineate colonies, provides a fully automated, accurate, and precise method for characterizing the biological potential and functional potency of the cultured cells. Furthermore, extracted parameters in addition to colony number provide means of further characterization and sub-classification of colony level statistics. These parameters include, but are not limited to, cell/nuclear count, cell/nuclear density, colony morphology (shape and size parameters), secondary marker coverage, effective proliferation rates, and so forth (Fig. A1.2). In addition to human connective tissue progenitors (CTPs) derived from bone, bone marrow, cartilage, adipose tissue, muscle, periosteum, and synovium, this practice and technology has been implemented in the cell and colony identification and characterization of several cell and tissue types including: umbilical cord blood hematopoietic stem cells (Fig. X1.2); adipose-derived stem cells (Fig. X1.3); and human epidermal (Fig. X1.4) and dermal (Fig. X1.5) stem cells.4.3 Benefits of Automated Analysis of CFU Assays—Automated analysis is expected to provide more rapid, reproducible, and precise results in comparison to the manual enumeration of cells and colonies utilizing a microscope and hemocytometer. In addition to being time consuming, labor intensive, and subjective, manual enumeration has been shown to have a significant degree of intra- and inter-observer variability, with coefficients of variation (CV) ranging from 8.1 % to 40.0 % and 22.7 % to 80 %, respectively. Standard CVs for cell viability assessment and progenitor (colony) type enumeration have been shown to range from 19.4 % to 42.9 % and 46.6 % to 100 %, respectively (4, 11, 12). In contrast, studies focusing on bacteria, bone marrow-derived stem cells and osteogenic progenitor cells have collectively concluded that automated enumeration provides significantly greater accuracy, precision, and/or speed for counting and sizing cells and colonies, relative to conventional manual methodologies (4-6). Automated methods for enumerating cells and colonies are less biased, less time consuming, less laborious, and provide greater qualitative and quantitative data for intrinsic characteristics of cell and colony type and morphology.4.4 Selection of Cell Culture Surface Area and Optimal Cell Seeding Density—When performing a CFU assay, optimizing the cell culture surface area and cell seeding density is critical to developing methods for generating reliable and reproducible colony- and cell-level data. If seeding density is too low, then the frequency of observed colonies is decreased. This can result in a sampling size that is inadequate to characterize the population of CFUs in the sample. If seeding density is too high, the colonies that are formed may be too closely spaced. Overlapping colony footprints compromise colony counting and characterization. Because the intrinsic range of CFU prevalence in a given cell source may vary widely, in many cases, a trial and error approach to optimizing cell seeding density (or range of densities) that are needed for a given cell source will be necessary. It is important to note that the more heterogeneous the cell source (for example, bone marrow), the more colonies that are needed to accurately represent the stem and progenitor cell constituents. Further, the cell type, effective proliferation rate (EPR) and specific cell culture conditions (for example, media, serum, factors, oxygen tension, and so forth) can impact colony formation. For example, the automated CFU assay depicted in Fig. A1.2 employs a six-day culture period, two media changes, 20 % oxygen tension, alpha-MEM media (with 25 % fetal bovine serum, ascorbate, dexamethasone and streptomycin), an optimized cell seeding density of 250 000 nucleated cells per cm2 (250 000 cells per 1 mL of cell culture medium) and a cell culture surface area of 22 mm by 22 mm (dual-chamber Lab-Tek culture slides) (12, 13).4.5 Useful Documents—A number of useful documents are available that address best practices for conducting quantitative measurements of cells using imaging approaches: Guide F2998, Guide F3294, ISO 20391-1, ISO 20391-2, and “FDA Guidance on Technical Performance Assessment of Digital Pathology Whole Slide Imaging Devices,” (14).1.1 This practice, provided its limitations are understood, describes a procedure for quantitative measurement of the number and biological characteristics of colonies derived from a stem cell or progenitor population using image analysis.1.2 This practice is applied in an in vitro laboratory setting.1.3 This practice utilizes: (a) standardized protocols for image capture of cells and colonies derived from in vitro processing of a defined population of starting cells in a defined field of view (FOV), and (b) standardized protocols for image processing and analysis.1.4 The relevant FOV may be two-dimensional or three-dimensional, depending on the CFU assay system being interrogated.1.5 The primary unit to be used in the outcome of analysis is the number of colonies present in the FOV. In addition, the characteristics and sub-classification of individual colonies and cells within the FOV may also be evaluated, based on extant morphological features, distributional properties, or properties elicited using secondary markers (for example, staining or labeling methods).1.6 Imaging methods require that images of the relevant FOV be captured at sufficient resolution to enable detection and characterization of individual cells and over a FOV that is sufficient to detect, discriminate between, and characterize colonies as complete objects for assessment.1.7 Image processing procedures applicable to two- and three-dimensional data sets are used to identify cells or colonies as discreet objects within the FOV. Imaging methods may be optimized for multiple cell types and cell features using analytical tools for segmentation and clustering to define groups of cells related to each other by proximity or morphology in a manner that is indicative of a shared lineage relationship (that is, clonal expansion of a single founding stem cell or progenitor).1.8 The characteristics of individual colony objects (cells per colony, cell density, cell size, cell distribution, cell heterogeneity, cell genotype or phenotype, and the pattern, distribution and intensity of expression of secondary markers) are informative of differences in underlying biological properties of the clonal progeny.1.9 Under appropriately controlled experimental conditions, differences between colonies can be informative of the biological properties and underlying heterogeneity of colony founding cells (CFUs) within a starting population.1.10 Cell and colony area/volume, number, and so forth may be expressed as a function of cell culture area (square millimeters), or initial cell suspension volume (milliliters).1.11 Sequential imaging of the FOV using two or more optical methods may be valuable in accumulating quantitative information regarding individual cells or colony objects in the sample. In addition, repeated imaging of the same sample will be necessary in the setting of process tracking and validation. Therefore, this practice requires a means of reproducible identification of the location of cells and colonies (centroids) within the FOV area/volume using a defined coordinate system.1.12 To achieve a sufficiently large field-of-view (FOV), images of sufficient resolution may be captured as multiple image fields/tiles at high magnification and then combined together to form a mosaic representing the entire cell culture area.1.13 Cells and tissues commonly used in tissue engineering, regenerative medicine, and cellular therapy are routinely assayed and analyzed to define the number, prevalence, biological features, and biological potential of the original stem cell and progenitor population(s).1.13.1 Common applicable cell types and cell sources include, but are not limited to: mammalian stem and progenitor cells; adult-derived cells (for example, blood, bone marrow, skin, fat, muscle, mucosa) cells, fetal-derived cells (for example, cord blood, placental/cord, amniotic fluid); embryonic stem cells (ESC) (that is, derived from inner cell mass of blastocysts); induced pluripotent cells (iPC) (for example, reprogrammed adult cells); culture expanded cells; and terminally differentiated cells of a specific type of tissue.1.13.2 Common applicable examples of mature differentiated phenotypes which are relevant to detection of differentiation within and among clonal colonies include: hematopoietic phenotypes (erythrocytes, lymphocytes, neutrophiles, eosinophiles, basophiles, monocytes, macrophages, and so forth), adult tissue-specific progenitor cell phenotypes (oteoblasts, chondrocytes, adipocytes, and so forth), and other tissues (hepatocytes, neurons, endothelial cells, keratinocyte, pancreatic islets, and so forth).1.14 The number of stem cells and progenitor cells in various tissues can be assayed in vitro by liberating the cells from the tissues using methods that preserve the viability and biological potential of the underlying stem cell and/or progenitor population, and placing the tissue-derived cells in an in vitro environment that results in efficient activation and proliferation of stem and progenitor cells as clonal colonies. The true number of stem cells and progenitors (true colony forming units (tCFU)) can thereby be estimated on the basis of the number of colony-forming units observed (observed colony forming units (oCFU)) to have formed (1-3)2 (Fig. A1.1). The prevalence of stem cells and/or progenitors can be estimated on the basis of the number of observed colony-forming units (oCFU) detected, divided by the number of total cells assayed.1.15 The automated image acquisition and analysis approach (described herein) to cell and colony enumeration has been validated and found to provide superior accuracy and precision when compared to the current “gold standard” of manual observer defined visual cell and colony counting under a brightfield or fluorescent microscope with or without a hemocytometer (4), reducing both intra- and inter-observer variation. Several groups have attempted to automate this and/or similar processes in the past (5, 6) . Recent reports further demonstrate the capability of extracting qualitative and quantitative data for colonies of various cell types at the cellular and even nuclear level (4, 7).1.16 Advances in software and hardware now broadly enable systematic automated analytical approaches. This evolving technology creates the need for general agreement on units of measurement, nomenclature, process definitions, and analytical interpretation as presented in this practice.1.17 Standardized methods for automated CFU analysis open opportunities to enhance the value and utility of CFU assays in several scientific and commercial domains:1.17.1 Standardized methods for automated CFU analysis open opportunities to advance the specificity of CFU analysis methods though optimization of generalizable protocols and quantitative metrics for specific cell types and CFU assay systems which can be applied uniformly between disparate laboratories.1.17.2 Standardized methods for automated CFU analysis open opportunities to reduce the cost of colony analysis in all aspects of biological sciences by increasing throughput and reducing work flow demands.1.17.3 Standardized methods for automated CFU analysis open opportunities to improve the sensitivity and specificity of experimental systems seeking to detect the effects of in vitro conditions, biological stimuli, biomaterials and in vitro processing steps on the attachment, migration, proliferation, differentiation, and survival of stem cells and progenitors.1.18 Limitations are described as follows:1.18.1 Colony Identification—Cell Source/Colony Type/Marker Variability—Stem cells and progenitors from various tissue sources and in different in vitro environments will manifest different biological features. Therefore, the specific means to detect cells or nuclei and secondary markers utilized and the implementation of their respective staining protocols will differ depending on the CFU assay system, cell type(s) and markers being interrogated. Optimized protocols for image capture and image analysis to detect cells and colonies, to define colony objects and to characterize colony objects will vary depending on the cell source being utilized and CFU system being used. These protocols will require independent optimization, characterization and validation in each application. However, once defined, these can be generalized between labs and across clinical and research domains.1.18.2 Instrumentation-Induced Variability in Image Capture—Choice of image acquisition components described above may adversely affect segmentation of cells and subsequent colony identification if not properly addressed. For example, use of a mercury bulb rather than a fiber-optic fluorescent light source or the general misalignment of optics could produce uneven illumination or vignetting of tiled images comprising the primary large FOV image. This may be corrected by applying background subtraction routines to each tile in a large FOV image prior to tile stitching.1.18.3 CFU Assay System Associated Variation in Imaging Artifacts—In addition to the presentation of colony objects with unique features that must be utilized to define colony identification, each image from each CFU system may present non-cell and non-colony artifacts (for example, cell debris, lint, glass aberrations, reflections, autofluorescence, and so forth) that may confound the detection of cells and colonies if not identified and managed.1.18.4 Image Capture Methods and Quality Control Variation—Variation in image quality will significantly affect the precision and reproducibility of image analysis methods. Variation in focus, illumination, tile registration, exposure time, quenching, and emission spectral bleeding, are all important potential limitations or threats to image quality and reproducibility.1.19 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.20 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.1.21 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 This plate format is useful for the routine monitoring of culturable, waterborne bacteria in potable and non-potable waters. The significance of finding these bacteria can help with identifying water quality or water system problems or evaluate compliance with maintenance protocols. This test method uses small volumes of water, or dilutions thereof, and provides an easy and reliable method that eliminates media preparation and reduces laboratory waste.1.1 This test method describes a simple procedure for the quantification of culturable, waterborne bacteria in potable water (drinking water, bottled water, and dental water, for example) and non-potable waters (cooling towers, for example).1.1.1 The EasyDisc2, 3 plate format is designed to test 1 mL of a water sample on a 47 mm gridded plate containing a growth reagent embedded to the plate’s inner surface.1.1.2 Detection is based on colorimetric technology in which viable, aerobic, heterotrophic, waterborne bacteria grow when present in the water sample, displaying a color reaction which allows for a simplified visualization of colony growth.1.2 Each plate can accurately detect up to 300 colony forming units per 1 mL (CFU/1 mL) of sample. To increase the quantification range, a sample dilution can be used. Adjust the CFU/mL result to reflect dilutions.1.3 This test method can be used for potable (for example, drinking, bottled, and dental) waters and non-potable waters such as cooling tower waters. It is the user’s responsibility to adhere to all requirements by local regulations and ensure the validity of this test method for waters other than those tested as part of the Interlaboratory Study (ILS).1.4 The values stated in SI units are to be regarded as the standard. No other units of measurement are included in this standard.1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 In-vitro osteoblast differentiation assays are one approach to screen progenitor stem cells for their capability to become osteoblasts. The extent of calcium deposits or mineralized matrix that form in vitro may be an indicator of differentiation to a functional osteoblast; however, expression of osteogenic genes or proteins is another important measurement to use in conjunction with this assay to determine the presence of an osteoblast.5.2 This practice provides a technique for staining, imaging, and quantifying the fluorescence intensity and area related to the mineralization in living cell cultures using the non-toxic calcium-chelating dye, XO. The positively stained area of mineralized deposits in cell cultures is an indirect measure of calcium content. It is important to measure the intensity to ensure that the images have not been underexposed or overexposed. Intensity and area do not correlate directly to calcium content.5.3 XO enables the monitoring of calcium deposits repeatedly throughout the life of the culture without detriment to the culture. There is no interference on subsequent measurements of the mineralized area due to dye accumulation from repeated application (1).3 Calcium deposits that have been previously stained may appear brighter, but this does not impact the area measurement. Calcein dyes may also be used for this purpose (1) but require a different procedure for analysis than XO (that is, concentration and filter sets) and are thus not included here. Alizarin Red and Von Kossa are not suitable for use with this procedure on living cultures since there is no documentation supporting their repeated use in living cultures without deleterious effects.5.4 The practice may be applied to cultures of any cells capable of producing calcium deposits. It may also be used to document the absence of mineral in cultures where the goal is to avoid mineralization.5.5 During osteoblast differentiation assays, osteogenic supplements are provided to induce or assist with the differentiation process. If osteogenic supplements are used in excess, a calcium deposit that is not osteoblast-mediated and is referred to as dystrophic, pathologic, or artifactual may occur in the cell cultures (2). For example, when higher concentrations of beta-glycerophosphate are used in the medium to function as a substrate for the enzyme alkaline phosphatase secreted by the cells, there is a marked increase in free phosphate, which then precipitates with Ca++ ions in the media to form calcium phosphate crystals independently of the differentiation status of the progenitor cell. Alkaline phosphatase production is associated with progenitor cell differentiation, and is frequently stimulated by dexamethasone addition to the medium, which enhances the formation of calcium deposits. These kinds of calcium/mineral deposits are thus considered dystrophic, pathologic, or artifactual because they were not initiated by a mature osteoblast. The measurement obtained by using this practice may thus result in a potentially false interpretation of the differentiation status of osteoprogenitor cells if used in isolation without gene or protein expression data (3, 4).5.6 Due to the possibility of artifactual calcium deposits during mineralization assays (2-4), gene expression analysis or protein analysis techniques demonstrating the RNA message or the presence of osteocalcin and bone sialoprotein are recommended for use in conjunction with the calcium deposit quantification procedure described here in order to confirm the presence of mature osteoblasts that are in the process of secreting a mineralizing matrix.5.7 The deposition of a mineralized substance in the culture dish does not confirm that the cells being cultured are capable of forming bone in vivo.5.8 The pattern of mineralized matrix deposition in the culture dish will vary, depending on the number of times the cells have been passaged (that is, first passage primary cells versus cells that have been passaged several times, including cell lines). First passage primary cells typically form relatively large nodules of osteoprogenitor cells that differentiate and mineralize, while cells that have been passaged many times lead to the formation of diffuse, dispersed mineral throughout the culture dish. This practice is independent of the pattern of mineralization and can be used to analyze mineralized matrix in both primary cells and cell lines.5.9 Since some cells proliferate slower than others and since some of the cell culture surfaces being tested may affect proliferation of the cells, the data can be normalized to total cell number. Since reduced proliferation typically reduces mineralization, normalization to cell number typically does not influence the outcomes. Total DNA content can be determined as an indirect measure of cell number. There are several commercially available kits for this purpose. Since DNA analysis is a destructive, toxic assay, additional cell cultures must be prepared if this assay is used.1.1 This practice defines a method for the estimation of calcium content at multiple time points in living cell cultures that have been cultured under conditions known to promote mineralization. The practice involves applying a fluorescent calcium-chelating dye that binds to the calcium phosphate mineral crystals present in the live cultures followed by image analysis of fluorescence microscopy images of the stained cell cultures. Quantification of the positively stained areas provides a relative measure of the calcium content in the cell culture plate. A precise correlation between the image analysis parameters and calcium content is beyond the scope of this practice.1.2 Calcium deposition in a secreted matrix is one of several features that characterize bone formation (in vitro and in vivo), and is therefore a parameter that may indicate bone formation and osteoblast function (that is, osteoblastic differentiation). Calcium deposition may, however, be unrelated to osteoblast differentiation status if extensive cell death occurs in the cell cultures or if high amounts of osteogenic medium components that lead to artifactual calcium-based precipitates are used. Distinguishing between calcium deposition associated with osteoblast-produced mineralized matrix and that from pathological or artifactual deposition requires additional structural and chemical characterization of the mineralized matrix and biological characterization of the cell that is beyond the scope of this practice.1.3 The parameters obtained by image analysis are expressed in relative fluorescence units or area percentage (area%), for example, fraction of coverage of the area analyzed.1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 This test method is intended to provide a tool for assessing whether fuel storage and distribution facilities or end user fuel tanks are subject to microbial growth and alert fuel suppliers or users to the potential for fuel quality or operational problems and/or the requirement for preventative or remedial measures.5.2 This test method detects numbers of microbial colony forming units (CFU), the same detection parameter used in the laboratory standard procedures Practice D6974 and IP 385. However, whereas Practice D6974 and IP 385 provide separate assessment of numbers of viable aerobic bacteria CFU and numbers of viable fungal CFU, this test method provides a combined total count of viable aerobic bacteria and fungal CFU.5.3 This test method is designed to detect a recognized group of microorganisms of significance in relation to contamination of distillate fuels, but it is recognized that microbiological culture techniques do not detect all microorganisms that can be present in a sample. Culturability is affected primarily by the ability of captured microbes to proliferate on the growth medium provided, under specific growth conditions. Consequently, a proportion of the active or inactive microbial population present in a sample can be viable but not detected by any one culture test.7 In this respect, the test is indicative of the extent of microbial contamination in a sample ,and it is assumed that when a fuel sample is significantly contaminated, some of the dominant microbial species present will be quantifiably detected, even if not all species present are culturable.5.4 Many samples from fuel systems can be expected to contain a low level of “background” microbial contamination, which is not necessarily of operational significance. The minimum detection level of this test method is determined by the volume of specimen tested and is set such that microbial contamination will generally only be detected when it is at levels indicative of active proliferation.5.5 The test will detect culturable bacteria and fungi that are metabolically active and dormant fungal spores. Presence of fungal spores in a fuel sample can be indicative of active microbial proliferation within a fuel tank or system, but at a point distant from the location sampled. Active microbial growth only occurs in free water, and this can be present only as isolated pockets at tank or system low points. Because fungal spores are more hydrophobic than active cells and fungal material (mycelium), they disperse more readily in fuel phase and are thus more readily detected when low points cannot be directly sampled and only fuel phase is present in samples.5.6 This test method can determine whether microbial contamination in samples drawn from fuel tanks and systems is absent or present at light, moderate, and heavy levels.5.7 The categorization of light, moderate, and heavy levels of contamination will depend on the fuel type, the sampling location, the facility sampled, and its specific operating circumstances.5.8 Further guidance or interpretation of test results can be found in Guide D6469, in the Energy Institute Guidelines for the investigation of the microbial content of petroleum fuels, and for the implementation of avoidance and remedial strategies and in the IATA Guidance Material on Microbiological Contamination in Aircraft Fuel Tanks.5.8.1 Further guidance on sampling can be found in Practice D7464.5.9 Testing can be conducted on a routine basis or to investigate incidents.5.10 Microbiological tests are not intended to be used to determine compliance with absolute fuel specifications or limits. The implementation of specification limits for microbiological contamination in fuels is generally not appropriate, and microbial contamination levels cannot be used alone or directly to make inferences about fuel quality or fitness for use.5.11 When interpreting results, it must be appreciated that the test result applies only to the specific sample and specimen tested and not necessarily to the bulk fuel. Microbiological contamination usually shows a highly heterogeneous distribution in fuel systems, and therefore, analysis of a single sample will rarely provide a complete assessment of the overall levels of contamination present.5.12 Water phase will usually contain substantially higher numbers of microbial CFU than fuel phase and, consequently, a different interpretation of results is required.1.1 This test method describes a procedure that can be used in the field or in a laboratory to quantify culturable, viable aerobic microorganisms present as contaminants in liquid fuels, including those blended with synthesized hydrocarbons or biofuels, with kinematic viscosities (at 40 °C) of ≤24 mm2 s-1 and heavy and residual fuels with kinematic viscosities (at 40 °C) of ≤700 mm2 s-1 and in fuel-associated water.1.1.1 This test method has been validated by an ILS for a range of middle distillate fuels meeting Specifications D975, D1655, ISO 8217 DMA, and NATO F-76.21.2 This test method quantitatively assesses culturable, viable aerobic microbial content present in the form of bacteria, fungi, and fungal spores. Results are expressed as the total number of microbial colony forming units (CFU)/L of fuel or total number of CFU/mL of associated water. The number of CFU should not be interpreted as absolute values but should be used as part of a diagnostic or condition monitoring effort; for example, these values can be used to assess contamination as absent, light, moderate, or heavy.NOTE 1: This test method is technically equivalent to IP 613, although the two methods are not currently jointed.1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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This specification covers the requirements for disposable glass culture tubes for use in culturing applications. Disposable glass culture tubes shall be made of type I (borosilicate) or type II (soda-lime) glass. Tube design shall be of one-piece construction. Other requirements shall include top or open end finish requirement, bottom or closed end requirement, residual thermal stress requirement, workmanship, and tube dimensions.1.1 This specification covers the requirements for disposable glass tubes suitable for general testing and culturing applications in blood banks, hematology, bacteriology, virology, and tissue culture laboratories.1.2 For practical purposes, the word “disposable” according to this specification and expected product performance expressed in this specification describes those disposable glass culture tubes that are to be used one time only. Any institution or individual who reuses a disposable glass culture tube must bear full responsibility for its safety and effectiveness.1.3 For packaging standards, choose among the following: Specifications E920 or E921 or Practice E1133.1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 The presence of cell growth medium complicates a direct analysis of cells with SIMS. Attempts to wash out the nutrient medium results in the exposure of cells to unphysiological reagents that may also alter their chemical composition. This obstacle is overcome by using a sandwich freeze-fracture method (1). This cryogenic method has provided a unique way of sampling individual cells in their native state for SIMS analysis.5.2 The procedure described here has been successfully used for imaging Na+ and K+ ion transport (3), calcium alterations in stimulated cells (4,5), and localization of therapeutic drugs and isotopically labeled molecules in single cells (6). The frozen freeze-dried cells prepared according to this method have been checked for SIMS matrix effects (7). Ion image quantification has also been achieved in this sample type (8).5.3 The procedure described here is amenable to a wide variety of cell cultures and provides a way for studying the response of individual cells for chemical alterations in the state of health and disease and localization of isotopically-labeled molecules and theraputic drugs in cell culture models.1.1 This guide provides the Secondary Ion Mass Spectrometry (SIMS) analyst with a cryogenic method for analyzing individual tissue culture cells growing in vitro. This guide is suitable for frozen-hydrated and frozen-freeze-dried sample types. Included are procedures for correlating optical, laser scanning confocal and secondary electron microscopies to complement SIMS analysis.1.2 This guide is not suitable for cell cultures that do not attach to the substrate.1.3 This guide is not suitable for any plastic embedded cell culture specimens.1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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4.1 This practice is useful for assessing cytotoxic potential both when evaluating new materials or formulations for possible use in medical applications, and as part of a quality control program for established medical materials and medical devices.4.2 This practice assumes that assessment of cytotoxicity potential provides one method for predicting the potential for cytotoxic or necrotic reactions to medical materials and devices during clinical applications to humans. In general, cell culture testing methods have shown good correlation with animal assays when only chemical toxicities are being considered.NOTE 1: The results obtained using this method may not predict in vivo behavior which can be influenced by multiple factors such as those arising from site of application or physical properties that may result from design and fabrication.4.3 This cell culture test method is suitable for adoption in specifications and standards for materials for use in the construction of medical devices that are intended to have direct contact with tissue, tissue fluids, or blood. However, care should be taken when testing materials that are absorbable, include an eluting or degradable coating, are liquid or gelatinous in nature, are irregularly shaped solid materials, or have a high density or mass, to make sure that the method is applicable. If leachables from the test sample are capable of diffusing through the agar layer, agarose-based methods such as Test Method F895 may be considered as an alternate method, depending on sample characteristics, or in cases where investigators wish to further evaluate the cytotoxic response of cells underlying the test sample.1.1 This practice covers a reference method of direct contact cell culture testing which may be used in evaluating the cytotoxic potential of materials for use in the construction of medical materials and devices.1.2 This practice may be used either directly to evaluate materials or as a reference against which other cytotoxicity test methods may be compared.1.3 This is one of a series of reference test methods for the assessment of cytotoxic potential, employing different techniques.1.4 Assessment of cytotoxicity is one of several tests employed in determining the biological response to a material, as recommended in Practice F748.1.5 The L-929 cell line was chosen because it has a significant history of use in assays of this type. This is not intended to imply that its use is preferred; only that the L-929 is a well characterized, readily available, established cell line that has demonstrated reproducible results in several laboratories.1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 This guide should be used by producers and potential producers of non-culture tests to determine the accuracy, selectivity, specificity, and precision of the tests, as defined in Practice E691. Results of such studies should identify the limitations and indicate the utility or applicability of the non-culture test, or both, for use on different types of samples. Guide E1488 recommends other statistical tools for evaluating the suitability and applicability of proposed new test methods.5.2 Non-culture test users and potential users should employ this guide to evaluate results of the non-culture test as compared to their present methods. Practices D5245 and D5465 should be reviewed in regards to the microbiological methods employed. If culture methods have not been used for monitoring the systems, then guidelines are included for obtaining microbiological expertise.5.3 Utilization of a non-culture test can reduce the time required to determine the microbiological status of the system and detect microbe that are not detected by culture testing. Consequently, non-culture tests can contribute to the improvement in the overall operating efficiency of microbial contamination condition monitoring and diagnostic efforts, and microbicide performance evaluations.5.4 Detecting microbial contamination levels that exceed predetermined upper control limits indicates the need for an addition of an antimicrobial agent or other corrective maintenance action. By accurately determining this in a shorter time period than is possible than by culture methods, treatment with antimicrobial agents may circumvent more serious problems than if the treatment were postponed until culture results were available. If the antimicrobial treatment program relied on an inaccurate non-culture test, then unnecessary loss of product and problems associated with inappropriate selection or improper dosing with antimicrobial agents would exist.5.5 Since many methods based on entirely different chemical and microbiological principles are considered, it is not possible to establish a unique design and recommend a specific method of statistical analyses for the comparisons to be made. It is only possible to present guides that should be followed while performing the experiments. It is also recommended that a statistician be involved in the study.1.1 The purpose of this guide is to assist users and producers of non-culture microbiological tests in determining the applicability of the test for processing different types of samples and evaluating the accuracy of the results. Culture test procedures such as the Heterotrophic (Standard) Plate Count, the Most Probable Number (MPN) method and the Spread Plate Count are widely cited and accepted for the enumeration of microorganisms. However, these methods have their limitations, such as performance time. Moreover, any given culture test method typically recovers only a portion of the total viable microbes present in a sample. It is these limitations that have recently led to the marketing of a variety of non-culture procedures, test kits and instruments.1.2 Culture test methods estimate microbial population densities based on the ability of mircoorganisms in a sample to proliferate in or on a specified growth medium, under specified growth conditions. Non-culture test methods attempt to provide the same or complimentary information through the measurement of a different parameter. This guide is designed to assist investigators in assessing the accuracy and precision of non-culture methods intended for the determination of microbial population densities or activities.1.3 It is recognized that the Heterotrophic Plate Count (HPC) does not recover all microorganisms present in a product or a system (1, 2).2 When this problem occurs during the characterization of a microbiological population, alternative standard enumeration procedures may be necessary, as in the case of sulfate-reducing bacteria. At other times, chemical methods that measure the rates of appearance of metabolic derivatives, the utilization of contaminated product components or genetic profile of the microbial population might be indicated. In evaluating non-culture tests, it is possible that the use of these alternative standard procedures might be the only means available for establishing correlation. In such cases, this guide can serve as a reference for those considerations.1.4 Because there are so many types of tests that could be considered non-culture based, it is impossible to recommend a specific test protocol with statistical analyses for evaluating the tests. Instead, this guide should assist in determining what types of tests should be considered to verify the utility and identify the limitations of the nonconventional test.1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 Biodeteriogenic microbes infecting fuel systems typically are most abundant within slime accumulations on system surfaces or at the fuel-water interface (Guide D6469). However, it is often impractical to obtain samples from these locations within fuel systems. Although the numbers of viable bacteria and fungi recovered from fuel-phase samples are likely to be several orders of magnitude smaller than those found in water-phase samples, fuel-phase organisms are often the most readily available indicators of fuel and fuel system microbial contamination.5.2 Growth Medium Selectivity—Guide E1326 discusses the limitations of growth medium selection. Any medium selected will favor colony formation by some species and suppress colony formation by others. As noted in 6.3, physical, chemical and physiological variables can affect viable cell enumeration test results. Test Method D7463 provides a non-culture means of quantifying microbial biomass in fuels and fuel associated water.5.3 Since a wide range of sample sizes, or dilutions thereof, can be analyzed by the membrane filter technique (Test Methods D5259 and F1094), the test sensitivity can be adjusted for the population density expected in the sample.5.4 Enumeration data should be used as part of diagnostic efforts or routine condition monitoring programs. Enumeration data should not be used as fuel quality criteria.1.1 This practice covers a membrane filter (MF) procedure for the detection and enumeration of Heterotrophic bacteria (HPC) and fungi in liquid fuels with kinematic viscosities ≤24 mm2 · s-1 at ambient temperature.1.2 This quantitative practice is drawn largely from IP Method 385 and Test Method D5259.1.3 This test may be performed either in the field or in the laboratory.1.4 The ability of individual microbes to form colonies on specific growth media depends on the taxonomy and physiological state of the microbes to be enumerated, the chemistry of the growth medium, and incubation conditions. Consequently, test results should not be interpreted as absolute values. Rather they should be used as part of a diagnostic or condition monitoring effort that includes other test parameters, in accordance with Guide D6469.1.5 This practice offers alternative options for delivering fuel sample microbes to the filter membrane, volumes or dilutions filtered, growth media used to cultivate fuel-borne microbes, and incubation temperatures. This flexibility is offered to facilitate diagnostic efforts. When this practice is used as part of a condition monitoring program, a single procedure should be used consistently.1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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4.1 This test method is useful for assessing the cytotoxic potential of new materials and formulations and as part of a quality control program for established medical devices and components.4.2 This test method assumes that assessment of cytotoxicity provides useful information to aid in predicting the potential clinical applications in humans. Cell culture methods have shown good correlation with animal assays and are frequently more sensitive to cytotoxic agents.4.3 This cell culture test method is suitable for incorporation into specifications and standards for materials to be used in the construction of medical devices that are to be implanted into the human body or placed in contact with tissue fluids or blood on a long-term basis.4.4 Some biomaterials with a history of safe clinical use in medical devices are cytotoxic. This test method does not imply that all biomaterials must pass this assay to be considered safe for clinical use (Practice F748).1.1 This test method is appropriate for materials in a variety of shapes and for materials that are not necessarily sterile. This test method would be appropriate in situations in which the amount of material is limited. For example, small devices or powders could be placed on the agar and the presence of a zone of inhibition of cell growth could be examined.1.1.1 This test method is not appropriate for leachables that do not diffuse through agar or agarose.1.1.2 While the agar layer can act as a cushion to protect the cells from the specimen, there may be materials that are sufficiently heavy to compress the agar and prevent diffusion or to cause mechanical damage to the cells. This test method would not be appropriate for these materials.1.2 The L-929 cell line was chosen because it has a significant history of use in assays of this type. This is not intended to imply that its use is preferred, only that the L-929 is an established cell line, well characterized and readily available, that has demonstrated reproducible results in several laboratories.1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

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