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2.1 The CHO/HGPRT assay detects forward mutations of the X-linked hypoxanthine-guanine phosphoribosyl transferase (hgprt) locus (coding for the enzyme, HGPRT) in Chinese hamster ovary (CHO) cells. Cells originally derived from Chinese hamster ovary tissue are exposed to a test article and, following an appropriate cell culture regimen, descendants of the original treated population are monitored for the loss of functional HGPRT, presumably due to mutations. Resistance to a purine analogue, 6-thioguanine (6TG) (or less desirably, 8-azaguanine (8AG)), is employed as the genetic marker. HGPRT catalyzes the conversion of the nontoxic 6TG to its toxic ribophosphorylated derivative. Loss of the enzyme or its activity therefore leads to cells resistant to 6TG.2.2 Because HGPRT is an enzyme of the purine nucleotide salvage pathway, loss of the enzyme is not a lethal event. Different types of mutational events (base substitutions, frameshifts, deletions, some chromosomal type lesions, and so forth) should theoretically be detectable at the hgprt locus. The CHO/HGPRT assay has been used to study a wide range of mutagens, including radiations (2-4), and a wide variety of chemicals (1), and complex chemical mixtures (5).1.1 This guide highlights some of the more relevant biological concepts as they are currently understood, and summarizes the critical technical aspects for acceptable bioassay performances as they currently are perceived and practiced. The Chinese hamster ovary cell/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) assay (1) 2 has been widely applied to the toxicological evaluation of industrial and environmental chemicals.1.2 This guide concentrates on the practical aspects of cell culture, mutagenesis procedures, data analysis, quality control, and testing strategy. The suggested approach represents a consensus of the panel members for the performance of the assay. It is to be understood, however, that these are merely general guidelines and are not to be followed without the use of sound scientific judgement. Users of the assay should evaluate their approach based on the properties of the substances to be tested and the questions to be answered.1.3 Deviation from the guidelines based on sound scientific judgement should by no means invalidate the results obtained.1.4 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 A large number of industrial processes involve transfer and feeding of bulk solids, and the ability of such materials to flow in a controlled manner during these operations is critical to product quality.5.2 Direct shear cells are among the most important methods for measuring the flow properties of bulk solids in industrial applications for bulk solids handling.5.3 Direct shear cells have many advantages over simpler methods of measuring bulk solids flow properties, but their operation is more complex and the procedures for their use must be carefully controlled to produce accurate and reproducible data.5.4 The three most popular direct shear cell types are: Translational (D6128), Annular (D6773), and Rotational (D6682 and D7891).5.5 From shear cell data, a wide variety of parameters can be obtained, including the yield locus representing the shear stress to normal stress relationship at incipient flow, angle of internal friction, unconfined yield strength, cohesion, and a variety of related parameters such as the flow function.5.6 In addition, these three direct shear cells can be set up with wall coupons to measure wall friction.5.7 When the shear cell data are combined with unconfined yield strength, wall friction data, and bulk density data, they can be used for bin and hopper evaluation and design.1.1 This guide covers theory and principles for obtaining reliable and accurate bulk solids flow data using a direct shear cell. It includes characteristics and limitations of the three most popular direct shear cell types: Translational (D6128), Annular (D6773), and Rotational (D6682 and D7891).1.2 Units—The values stated in SI units are to be regarded as standard. No other units of measure are included in this standard.1.3 This guide offers an organized collection of information or a series of options and does not recommend a specific course of action. This document cannot replace education or experience and should be used in conjunction with professional judgment. Not all aspects of this guide may be applicable in all circumstances. This ASTM standard is not intended to represent or replace the standard of care by which the adequacy of a given professional service must be judged, nor should this document be applied without consideration of a project’s many unique aspects. The word “Standard” in the title of this document means only that the document has been approved through the ASTM consensus process.1.4 This guide offers an organized collection of information or a series of options and does not recommend a specific course of action. This document cannot replace education or experience and should be used in conjunction with professional judgment. Not all aspects of this guide may be applicable in all circumstances. This ASTM standard is not intended to represent or replace the standard of care by which the adequacy of a given professional service must be judged, nor should this document be applied without consideration of a project’s many unique aspects. The word “Standard” in the title of this document means only that the document has been approved through the ASTM consensus process.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 The test can be used to evaluate the following:5.1.1 Classification or Comparison of Powders—There are several parameters that can be used to classify powders relative to each other, the most useful being the measured shear stresses, cohesion, flow function and angle of internal friction.5.1.2 Sensitivity Analysis—The shear cell can be used to evaluate the relative effects of a range of powder properties and/or environmental parameters such as (but not limited to) humidity, particle size and size distribution, particle shape and shape distribution, moisture content and temperature.5.1.3 Quality Control—The test can, in some circumstances, be used to assess the flow properties of a raw material, intermediate or product against pre-determined acceptance criteria.5.1.4 Storage Vessel Design—Mathematical models exist for the determination of storage vessel design parameters which are based on the flow properties of powders as generated by shear cell testing, requiring shear testing at a range of consolidating stresses as well as the measurement of the wall friction angle with respect to the material of construction of the storage vessel. The methods are detailed in Refs. (1-3).2NOTE 1: The quality of the result produced by this test method is dependent on the competence of the personnel performing it, and the suitability of the equipment and facilities used. Agencies that meet the criteria of Practice D3740 are generally considered capable of competent and objective testing/sampling/inspection/etc. Users of this test method are cautioned that compliance with Practice D3740 does not in itself assure reliable results. Reliable results depend on many factors; Practice D3740 provides a means of evaluating some of those factors (4).Practice D3740 was developed for agencies engaged in the testing and/or inspection of soil and rock. As such it is not totally applicable to agencies performing this test method. However, users of this test method should recognize that the framework of Practice D3740 is appropriate for evaluating the quality of an agency performing this practice. Currently there is no known qualifying national authority that inspects agencies that perform this test method.1.1 This method covers the apparatus and procedures for measuring the incipient failure properties of a powder as a function of the normal stress for a given level of consolidation. The method also allows the further determination of the unconfined yield strength, internal friction angles, cohesion, flow function, major principal stress and wall friction angle (with the appropriate wall coupon fitted to the correct accessory).1.2 These parameters are most commonly used for the design of storage hoppers and bins using industry standard calculations and procedures. They can also provide relative classification or comparison of the flow behavior of different powders or different batches of the same powder if similar stress and shear regimes are encountered within the processing equipment.1.3 The apparatus is suitable for measuring the properties of powders with a maximum particle size of 1 mm. It is possible to test powders which have a small proportion of particles of 1 mm or greater, but they should be present in the bulk sample as no more than 5 % of the total mass in samples with a normal (Gaussian) size distribution.1.4 All observed and calculated values shall conform to the guidelines for significant digits and rounding established in Practice D6026.1.4.1 The procedures used to specify how data are collected/recorded or calculated, in this standard are regarded as the industry standard. In addition, they are representative of the significant digits that generally should be retained. The procedures used do not consider material variation, purpose for obtaining the data, special purpose studies, or any considerations for the user’s objectives; and it is common practice to increase or reduce significant digits of reported data to be commensurate with these considerations. It is beyond the scope of this standard to consider significant digits used in analysis methods for engineering design.1.5 Units—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 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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3.1 The electrical sensing zone method for cell counting is used in tissue culture, government research, and hospital, biomedical, and pharmaceutical laboratories for counting and sizing cells. The method may be applicable to a wide range of cells sizes and cell types, with appropriate validation (10).3.2 The electrical sensing zone methodology was introduced in the mid-1950s (9). Since this time, there have been substantial improvements which have enhanced the operator's ease of use. Among these are the elimination of the mercury manometer, reduced size, greater automation, and availability of comprehensive statistical computer programs.3.3 This instrumentation offers a rapid result as contrasted to the manual counting of cells using the hemocytometer standard counting chamber. The counting chamber is known to have an error of 10 to 30 %, as well as being time-consuming (11). In addition, when counting and sizing porcine hepatocytes, Stegemann et al concluded that the automated, electrical sensing zone method provided greater accuracy, precision, and speed, for both counts and size, compared to the conventional microscopic or the cell mass-based method (7).1.1 This test method, provided the limitations are understood, covers a procedure for both the enumeration and measurement of size distribution of most all cell types. The instrumentation allows for user-selectable cell size settings and is applicable to a wide range of cell types. The method works best for spherical cells, and may be less accurate if cells are not spherical, such as for discoid cells or budding yeast. The method is appropriate for suspension as well as adherent cell cultures (1).2 Results may be reported as number of cells per milliliter or total number of cells per volume of cell suspension analyzed. Size distribution may be expressed in cell diameter or volume.1.2 Cells commonly used in tissue-engineered medical products (2) are analyzed routinely. Examples are chondrocytes (3), fibroblasts (4), and keratinocytes (5). Szabo et al. used the method for both pancreatic islet number and volume measurements (6). In addition, instrumentation using the electrical sensing zone technology was used for both count and size distribution analyses of porcine hepatocytes placed into hollow fiber cartridge extracorporeal liver assist systems. In this study (7), and others (6, 8), the automated electrical sensing zone method was validated for precision when compared to the conventional visual cell counting under a microscope using a hemocytometer. Currently, it is not possible to validate cell counting devices for accuracy, since there not a way to produce a reference sample that has a known number of cells. The electrical sensing zone method shall be validated each time it is implemented in a new laboratory, it is used on a new cell type, or the cell counting procedure is modified.1.3 Electrical sensing zone instrumentation (commonly referred to as a Coulter counter) is manufactured by a variety of companies and is based upon electrical impedance. This test method, for cell counting and sizing, is based on the detection and measurement of changes in electrical resistance produced by a cell, suspended in a conductive liquid, traversing through a small aperture (see Fig. 1(9)). When cells are suspended in a conductive liquid, phosphate-buffered saline for instance, they function as discrete insulators. When the cell suspension is drawn through a small cylindrical aperture, the passage of each cell changes the impedance of the electrical path between two submerged electrodes located on each side of the aperture. An electrical pulse, suitable for both counting and sizing, results from the passage of each cell through the aperture. The path through the aperture, in which the cell is detected, is known as the “electronic sensing zone.” This test method permits the selective counting of cells within narrow size distribution ranges by electronic selection of the generated pulses. While the number of pulses indicates cell count, the amplitude of the electrical pulse produced depends on the cell's volume. The baseline resistance between the electrodes is due to the resistance of the conductive liquid within the boundaries of the aperture. The presence of cells within the “electronic sensing zone” raises the resistance of the conductive pathway that depends on the volume of the cell. Analyses of the behavior of cells within the aperture demonstrates that the height of the pulse produced by the cell is the parameter that most nearly shows proportionality to the cell volume.1.4 Limitations are discussed as follows:1.4.1 Coincidence—Occasionally, more than a single cell transverses the aperture simultaneously. Only a single larger pulse, as opposed to two individual pulses, is generated. The result is a lower cell count and higher cell volume measurement. The frequency of coincidence is a statistically predictable function of cell concentration that is corrected by the instrument. This is called coincidence correction (8). This phenomenon may be reduced by using lower cell concentrations.1.4.2 Viability—Electrical sensing zone cell counting enumerates both viable and nonviable cells and cannot determine percent viable cells. A separate test, such as Trypan blue, is required to determine percent viable cells.1.4.3 Cell Diameter—This is a function of the size range capability of the aperture size selected. Measurements may be made in the cell diameter range of 0.6 μm to 1200 μm. Setting the counting size range on the instrument can affect the test results, especially if the cell size has a large distribution, and should be carefully controlled to help achieve repeatability.1.4.4 Size Range of the Aperture—The size range for a single aperture is proportional to its diameter. The response has been found to depend linearly on diameter over a range from 2 % to 80 % of the diameter. However, the aperture tube may become prone to blockage at levels greater than 60 % of diameter. Therefore, the practical operating range of the aperture is considered to be 2 % to 60 % of the diameter.1.4.5 Humidity—10 % to 85 %.1.4.6 Temperature—10 °C to 35 °C.1.4.7 Electrolyte Solution—The diluent for cell suspension shall provide conductivity and have minimal effect on cell size. The electrolyte of choice is commonly phosphate-buffered saline.

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5.1 There are no reproducible standardized protocols for preparing specimens used to evaluate the microbicidal efficacy of non-chemical treatments such as ultraviolet (UV), highenergy electron beam, or other forms of non-chemical antimicrobial technologies.5.2 Conventional protocols for applying bioburdens to carriers (see Test Method E2197) cause cells to stack upon one another, thereby creating multiple cell layers in which cells in layers closer to the carrier are masked by cells in overlying layers, which makes relative comparison of different non-chemical antimicrobial treatments more difficult.5.3 Steel and other metal carriers have asperities that can shield a percentage of the applied cells from direct exposure to electromagnetic irradiation.5.4 The combined effects of 5.2 and 5.3 confound determination of the microbicidal effect of electromagnetic irradiation on test specimens.5.5 The practice addresses these two confounding factors by:5.5.1 Using glass microscope slides – the surfaces of which are asperity-free – as carriers.5.5.2 Reliably depositing bacterial cells onto the carrier as a monolayer.5.6 The resulting specimen ensures that all microbes deposited onto the carrier are exposed equally to the irradiation source thereby ensuring that the only variables are the controlled ones – starting inoculum concentration, wavelength (λ – in nm), exposure time(s), and resulting energy dose (J).1.1 This practice provides a protocol for creating bacterial cell monolayers on a flat surface.1.2 The cultures used and culture preparation steps in this Practice are similar to AOAC Method 961.02 and US EPA MB-06. However, test bacteria are applied to the carrier using an automated deposition device (6.2) rather than as a suspension droplet.1.3 The carrier inspection protocol is similar to US EPA MB-03 except that carrier surfaces are inspected microscopically rather than visually, unaided.1.4 A monolayer of cells eliminates the confounding effect caused by the shadowing effect of outer layers of bacteria stacked upon other bacteria on test specimens – thereby attenuating directed energy beams (that is, ultraviolet light, high-energy electron beams) before they can reach underlying cells.1.5 An asperity-free surface eliminates the shadowing effect of specimen surface topology that can block direct exposure of target bacteria to non-chemical antimicrobial treatments.1.6 This practice provides a reproducible target microbe and surface specimen to minimize specimen variability within and between testing facilities. This facilitates direct data comparisons among various non-chemical antimicrobial technologies.1.6.1 Antimicrobial pesticides used in clinical and industrial applications are expected to overcome shadowing effects. However, this practice meets a need for a protocol that facilitates relative comparisons among non-chemical antimicrobial treatments.1.6.2 This practice is not intended to satisfy or replace existing test requirements for liquid chemical antimicrobial treatments (for example Test Methods E1153 and E2197) or established regulatory agency performance standards such as US EPA MB-06.1.7 This practice was validated using Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 15442) using a protocol based on AOAC Method 961.02. If other cultures are used, the suitability of this practice must be confirmed by inspecting prepared surfaces, by using scanning electron microscopy (SEM) or comparable high-resolution microscopy.1.8 The specimens prepared in accordance with this practice are not meant to simulate end-use conditions.1.8.1 Non-chemical technologies are only to be used on visibly clean, non-porous surfaces. Consequently, a soil load is not used.1.9 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.10 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.1.11 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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