5.1 This practice allows for the recovery and enumeration of viable and culturable, non-tuberculosis, rapidly growing Mycobacteria (M. immunogenum, M. chelonae, M. absessus, M. fortuitum, and M. smegmatis) in the presence of high Gram-negative background populations in metalworking fluid field samples. This population is predominantly comprised of Gram-negative bacteria and fungi. Mycobacterial contamination of metalworking fluids has been putatively associated with hypersensitivity pneumonitis (HP) amongst metalgrinding machinists. The detection and enumeration of these organisms will aid in better understanding of occupational health-related problems and a better assessment of antimicrobial pesticide efficacy.5.2 The measurement of viable and culturable mycobacterial densities (Guide E1326), combined with the total mycobacterial counts (including viable culturable (VC), viable nonculturable (VNC) and nonviable (NV) counts), is usually the first step in establishing any possible relationship between Mycobacteria and occupational health concerns (for example, HP).5.3 The practice can be employed in survey studies to characterize the viable-culturable mycobacterial population densities of metalworking fluid field samples.5.4 This practice is also applicable for establishing the mycobacterial resistance of metalworking fluid formulations by determining mycobacterium survival by means of plate count technique.5.5 This practice can also be used to evaluate the relative efficacy of microbicides against Mycobacteria in metalworking fluids.1.1 This practice covers the detection and enumeration of viable and culturable rapidly growing Mycobacteria (RGM), or non-tuberculosis Mycobacteria (NTM) in aqueous metalworking fluids (MWF) in the presence of high non-mycobacterial background population using standard microbiological culture methods.1.2 The detection limit is one colony forming unit (CFU)/mL metalworking fluid.1.3 This practice involves culture of organisms classified as Level 2 pathogens, and should be undertaken by a trained microbiologist in an appropriately equipped facility. The microbiologist should also be capable of distinguishing the diverse colonies of Mycobacteria from other microorganism colonies on a Petri dish and capable of confirming Mycobacteria by acid-fast staining method.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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5.1 Measurement of mycobacterial cell count densities is an important step in establishing a possible relationship between mycobacteria and occupational health-related allergic responses, for example, hypersensitivity pneumonitis (HP) in persons exposed to aerosols of metalworking fluids. It is known that the viable mycobacteria count underestimates the total mycobacterial levels by not counting the non-culturable, possibly dead or moribund population that is potentially equally important in the investigation of occupational health-related problems. The direct microscopic counting method (DMC) described here gives a quantitative assessment of the total numbers of acid-fast bacilli. It involves using acid-fast staining to selectively identify mycobacteria from other bacteria, followed by enumeration or direct microscopic counting of a known volume over a known area. Although other microbes—particularly the Actinomycetes—also stain acid-fast, they are differentiated from the mycobacteria because of their morphology and size. Non-mycobacteria, acid-fast microbes are 50 to 100 times larger than mycobacteria. This practice provides quantitative information on the total (culturable and non-culturable viable, and non-viable) mycobacteria populations. The results are expressed quantitatively as mycobacteria per mL of metalworking fluid sample.5.2 The DMC method using the acid-fast staining technique is a semi-quantitative method with a relatively fast turnaround time.5.3 The DMC method can also be employed in field survey studies to characterize the changes in total mycobacteria densities of metalworking fluid systems over a long period of time.5.4 The sensitivity detection limit of the DMC method depends on the MF and the sample volume (direct or centrifuged, etc.) examined.1.1 This practice describes a direct microscopic counting method (DMC) for the enumeration of the acid-fast stained mycobacteria population in metalworking fluids. It can be used to detect levels of total mycobacteria population, including culturable as well as non-culturable (possibly dead or moribund) bacterial cells. This practice is recommended for all water-based metalworking fluids (Classification D2881).1.2 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. For additional safety information, see Laboratory Safety: Principle and Practices, 4th Edition.21.3 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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Measurement of bacterial densities is generally the first step in establishing a relationship between bacteria and other biochemical processes. It is known that the classical plate count procedure underestimates bacterial densities while the epifluorescence direct microscopic procedure more accurately depicts the total numbers of nonviable or dormant and viable cells in a water sample. The acridine-orange INT-formazan reduction technique provides information on the total concentrations of bacteria as well as that proportion which are actively respiring and thus involved in degradative processes.The acridine-orange INT-formazan reduction technique is both quantitative and precise.This procedure is ideal for enumerating both pelagic and epibenthic bacteria in all fresh water and marine environments.The process can be employed in survey studies to characterize the bacteriological densities and activities of environmental waters.1.1 This test method covers the detection and enumeration of aquatic bacteria by the use of an acridine-orange epifluorescence direct-microscopic counting procedure. This test method is applicable to environmental waters and potable waters.1.2 Certain types of debris and other microorganisms may fluoresce in acridine-orange stained smears.1.3 The procedure described requires a trained microbiologist or technician who is capable of distinguishing bacteria from other fluorescing bodies on the basis of morphology when viewed at higher magnifications.1.4 Use of bright light permits differentiation of single bacteria where reduced formazan is deposited at the polar ends.1.5 Approximately 104 cells/mL are required for detection by this test method.1.6 Minimal cell size which allows the detection of formazan deposits is represented by bacteria of 0.4 μm.21.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.8 This standard does not purport to address 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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5.1 Bacterial populations, as part of the microbial community in aquatic systems are actively involved in nutrient cycling. The significance of these populations is often difficult to ascertain because of the presence of many physiological types. However, measurement of bacterial densities is usually the first step in trying to establish any relationship that might exist between bacteria and other biochemical processes.45.2 Acridine-orange epifluorescence direct-counting procedure cannot differentiate between viable and nonviable cells.5.3 This procedure cannot be used to convert directly the numbers to total carbon biomass because of the natural variations in bacterial cell size.5.4 The acridine-orange epifluorescence direct-microscopic count is both quantitative and precise.5.5 This procedure is ideal for enumerating both pelagic and epibenthic bacteria in all fresh water and marine environments.55.6 The process can be employed in survey activities to characterize the bacteriological densities of environmental waters.5.7 The procedure can also be used to estimate bacterial densities in cooling tower waters, process waters, and waters associated with oil drilling wells.1.1 This test method describes a procedure for detection and enumeration of aquatic bacteria by the use of an acridine-orange epifluorescence direct-microscopic counting procedure. It is applicable to environmental waters.1.2 Certain types of debris and other microorganisms may fluoresce in acridine orange-stained smears.1.3 The test method requires a trained microbiologist or technician who is capable of distinguishing bacteria from other fluorescing bodies on the basis of morphology when viewed at higher magnifications.21.4 Use of bright light permits differentiation of single bacteria where reduced formazan is deposited at the polar ends.1.5 Approximately 104 cells/mL are required for detection by this test method.21.6 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 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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5.1 P. aeruginosa is an opportunistic pathogen and has been linked as the causative agent of numerous infections that may be transmitted through a contaminated water supply to a susceptible host.NOTE 1: Fecal waste is >95 % E. coli which is found in humans and warm bloodied animals.5.2 The membrane filtration procedure described is a rapid and reliable test method of detecting P. aeruginosa in water.1.1 The test method covers the isolation and enumeration of Pseudomonas aeruginosa. Testing was performed on spiked samples using reagent grade water as the diluent from surface waters; recreational waters; ground water, water supplies; especially rural nonchlorinated sources; waste water; and saline waters. The detection limit of this test method is one microorganism per 100 mL.1.2 This test method was used successfully with reagent water. It is the user's responsibility to ensure the validity of this test method for surface waters, recreational waters, ground water, rural nonchlorinated sources; waste water; and saline waters.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. Specific hazard statements are given in Section 10.1.5 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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5.1 The enterococci are indicators of the bacteriological quality for potable water, shellfish growing waters, ambient, and recreational waters. A direct relationship between swimming, associated gastroenteritis, and enterococci has been established through epidemiological studies and marine and fresh water bathing beaches. These studies have led to the development of criteria that can be used to establish bathing water standards based on established health-water quality relationships.5.2 Since small or large volumes of water or dilutions thereof, can be analyzed by the membrane filter technique, a wide range of levels of enterococci in water can be enumerated and detected.1.1 This test method covers a membrane filter (MF) procedure for the detection and enumeration of the enterococci bacteria in water. The enterococci, which include Entero-coccus faecalis (E. faecalis), E. faecium, and their varieties are commonly found in the feces of humans and other warm-blooded animals. Although some strains are ubiquitous and not related to fecal pollution, enterococci in water are an indication of fecal pollution and the possible presence of enteric pathogens. These bacteria are found in water and wastewater in a wide range of densities. The detection limit is one colony forming unit (CFU)/volume filtered.1.2 This test method has been used successfully with temperate fresh and marine ambient waters, and wastewaters. It is the user’s responsibility to ensure the validity of this test method for waters of untested types.1.3 The values stated in SI units are to be regarded as standard. The values given in parentheses after SI units are included for information only and are not considered 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. For specific hazard statements, see Section 9.1.5 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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C. albicans is a yeast that is found as a commensal in the gastrointestinal, genitourinary, and alimentary tracts of healthy individuals, both human and lower animals (3, 4, 5). As such, it is a serious opportunistic pathogen of humans and may cause superficial or deep mycotic infections. Consequently, the yeast is found in raw sewage and in natural waters receiving human and animal wastes. C. albicans can survive in situ in seawater for at least six days (6). In vitro survival of the yeast in distilled (7) and lake water (8) has been demonstrated also. While there is at present no epidemiological evidence connecting human disease caused by C. albicans and use of water, the organism may be a useful indicator of recreational water quality (9). The test method may be applied to the monitoring of various treatment processes for efficiency in removing particular pathogens in waste water prior to discharge in receiving waters which in turn may be used again for a variety of purposes. Both public health and sanitary engineering interests should be aware of the presence of this yeast in wastewater and the potential for disease in contiguous waters. Future studies between the incidence of C. albicans and traditional water quality indicators (for example, total and fecal coliforms, fecal streptococci) may reveal a correlation of value in the assessment of potential health risks of swimming or other recreational waters.1.1 This test method covers the detection and enumeration of the yeast Candida albicans in raw sewage, waste waters, and natural waters.1.2 It is the responsibility of the analyst to determine if this test method yields satisfactory results in waters of other matrices.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. For a specific hazard statement, see Section .

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Clostridium perfringens is a strict obligate anaerobe that is found in fecal material. Under moderately adverse conditions these organisms produce endospores that can withstand extreme environmental conditions and are conservative tracers of past and present pollution in fresh and marine waters and sediments.1.1 This test method can enumerate Clostridium perfringens spores and vegetative cells from marine water, sediment, wastewater, ambient water, and drinking water. Since C. perfringens spores are present in large numbers in human and animal wastes and are resistant to wastewater treatment practices, extremes in temperature, and environmental stress, they are an indicator of present fecal contamination as well as a conservative tracer of past fecal contamination. It is the user's responsibility to ensure the validity of this test method for waters of untested matrices.1.2 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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5.1 This test method is useful for measuring recreational water quality and chlorinated wastewaters, although it can be used for any water suspected of contamination by fecal wastes of warm-blooded animals. The significance of finding E. coli in recreational water samples, especially samples obtained from fresh recreational waters, is that there is a risk of gastrointestinal illness, directly related to the E. coli density, associated with swimming.55.2 Since small or large volumes of water or dilutions thereof can be analyzed by the MF technique, a wider range of levels of E. coli in water can be detected and enumerated than with other methods.1.1 This test method describes a membrane filter (MF) procedure for the detection and enumeration of Escherichia coli, a bacterium found exclusively in the feces of humans and other warm-blooded animals. The presence of these microorganisms in water is an indication of fecal pollution and the possible presence of enteric pathogens. These bacteria are found in water and wastewater in a wide range of densities. The detection limit of this procedure is one colony forming unit (CFU) per volume filtered.1.2 This test method has been used successfully with temperate fresh and marine ambient waters, and wastewaters. It is the user’s responsibility to ensure the validity of this test method for waters of other types.1.3 The values stated in SI units are to be regarded as 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. For specific hazard statements, see Section 9.1.5 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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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 enumerates salt tolerant (halophilic) bacteria, and proteolytic bacteria that are also salt tolerant. Under the conditions of this test method those bacteria are equated as halophilic organisms. Salt tolerant proteolytic bacteria have been known to cause damage to hides and skins in raceway brine.1.1 This test method covers the enumeration of bacteria that can tolerate high salt concentrations or can hydrolyze protein/collagen, or both. This test method is applicable to raceway brine, brine-cured hides and skins, and pre-charge raceway liquor.1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.3 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.1.4 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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4.1 This test method enumerates salt tolerant yeast and mold, and under the conditions of this test method those are equated as halophilic organisms. Salt tolerant yeast and mold have been known to cause damage to hides and skins in raceway brine.1.1 This test method covers the enumeration of yeast and mold. This test method is applicable to raceway brine, brine-cured hides and skins, and pre-charge raceway liquor.1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.3 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.1.4 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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4.1 This test method enumerates proteolytic bacteria. Proteolytic bacteria have been known to cause damage to hides and skins.1.1 This test method covers the enumeration of bacteria that can hydrolyze protein/collagen in fresh (uncured) hides and skins. This test method is applicable to uncured hides and skins.1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.3 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.1.4 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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4.1 This test method enumerates yeast and mold. Yeast and mold have been known to cause damage to hides and skins.1.1 This test method covers the enumeration of yeast and mold on fresh (uncured) hides and skins. This test method is applicable to uncured hides and skins.1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.3 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.1.4 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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