ASTM E2564-23
(Practice)Standard Practice for Enumeration of Mycobacteria in Metalworking Fluids by Direct Microscopic Counting (DMC) Method
Standard Practice for Enumeration of <emph type="ital">Mycobacteria</emph> in Metalworking Fluids by Direct Microscopic Counting (DMC) Method
SIGNIFICANCE AND USE
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.
SCOPE
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.2
1.3 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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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.
Designation: E2564 − 23 An American National Standard
Standard Practice for
Enumeration of Mycobacteria in Metalworking Fluids by
1
Direct Microscopic Counting (DMC) Method
This standard is issued under the fixed designation E2564; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope* 3.2 Definitions of Terms Specific to This Standard:
3.2.1 acid-fast bacteria, n—a distinctive staining property
1.1 This practice describes a direct microscopic counting
of Mycobacteria due to their lipid-rich cell walls.
method (DMC) for the enumeration of the acid-fast stained
3.2.1.1 Discussion—Once stained, mycobacteria resist de-
mycobacteria population in metalworking fluids. It can be used
colorization when exposed to acidified organic solvents, and
to detect levels of total mycobacteria population, including
are therefore informally designated acid-fast.
culturable as well as non-culturable (possibly dead or mori-
3.2.2 non-tuberculous Mycobacteria (NTM)—
bund) bacterial cells. This practice is recommended for all
environmental mycobacteria not associated with tuberculosis.
water-based metalworking fluids (Classification D2881).
3.2.3 microscopic factor (MF), n—a calibrated conversion
1.2 This standard does not purport to address all of the
factor for calculating the mycobacterium count per mL sample.
safety concerns, if any, associated with its use. It is the
3.2.3.1 Discussion—The average number of mycobacterium
responsibility of the user of this standard to establish appro-
cells per one microscopic field (or oil field, OIF) is multiplied
priate safety, health, and environmental practices and deter-
by the MF to give the concentration of mycobacteria per mL of
mine the applicability of regulatory limitations prior to use.
sample.
For additional safety information, see Laboratory Safety: Prin-
2
ciple and Practices, 4th Edition.
3.2.4 oil immersion field (OIF), n—the circular area of a
1.3 This international standard was developed in accor-
microscopic field visible in the eyepiece of the microscope
dance with internationally recognized principles on standard-
using oil immersion objective.
ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
4. Summary of Practice
mendations issued by the World Trade Organization Technical
4.1 This practice describes a semi-quantitative test for
Barriers to Trade (TBT) Committee.
enumerating acid-fast stained environmental mycobacteria
(AFB) from metalworking fluids by direct microscopic count-
2. Referenced Documents
4
3 ing (DMC) method. It is used to determine total mycobacte-
2.1 ASTM Standards:
rium counts, including culturable and possibly dead or mori-
D2881 Classification for Metalworking Fluids and Related
bund cells in the sample. This practice cannot be used to
Materials
determine the total viable mycobacterium population in the
E2523 Terminology for Metalworking Fluids and Opera-
sample. A known sample volume (centrifuged or direct) is
tions
2
spread over a known area (1 cm or similar) on a microscope
3. Terminology slide (marked by frosted or painted circles). Following differ-
5
ential acid-fast staining, the acid-fast cells are counted in
3.1 For definitions of terms used in this standard, refer to
several microscopic fields over the designated area. The
Terminology E2523.
calculation is based on using a calibrated microscope with a
known microscopic factor (MF). The MF is determined by the
1
This practice is under the jurisdiction of ASTM Committee E34 on Occupa-
microscopic area over which a known amount of sample was
tional Health and Safety and is the direct responsibility of Subcommittee E34.50 on
Health and Safety Standards for Metal Working Fluids.
spread, the number of microscopic fields in the marked circle,
Current edition approved Oct. 1, 2023. Published October 2023. Originally
approved in 2007. Last previous edition approved in 2018 as E2564 – 18. DOI:
10.1520/E2564-23.
2 4
Gilchrist, Mary J. R., “Biosafety Precautions for Airborne Pathogens,” in Standard Methods for the Examination of Dairy Products, Chapter: 10: Direct
Laboratory Safety: Principles and Practices, ASM Press, 1995, pp. 67–76. Microscopic Methods for Bacteria or Somatic Cells, 16th ed., American Public
3
For referenced ASTM standards, visit the ASTM website, www.astm.org, or Health Association, Inc., Washington, DC, 1978.
5
contact ASTM Customer Service at service@astm.org. F
...
This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E2564 − 18 E2564 − 23 An American National Standard
Standard Practice for
Enumeration of Mycobacteria in Metalworking Fluids by
1
Direct Microscopic Counting (DMC) Method
This standard is issued under the fixed designation E2564; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope Scope*
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
2
regulatory limitations prior to use. For additional safety information, see Laboratory Safety: Principle and Practices, 4th Edition.
1.3 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.
2. Referenced Documents
3
2.1 ASTM Standards:
D2881 Classification for Metalworking Fluids and Related Materials
E2523 Terminology for Metalworking Fluids and Operations
3. Terminology
3.1 For definitions of terms used in this standard, refer to Terminology E2523.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 acid-fast bacteria, n—a distinctive staining property of Mycobacteria due to their lipid-rich cell walls.
3.2.1.1 Discussion—
Once stained, mycobacteria resist decolorization when exposed to acidified organic solvents, and are therefore informally
designated acid-fast.
3.2.2 non-tuberculous Mycobacteria (NTM)—environmental mycobacteria not associated with tuberculosis.
1
This practice is under the jurisdiction of ASTM Committee E34 on Occupational Health and Safety and is the direct responsibility of Subcommittee E34.50 on Health
and Safety Standards for Metal Working Fluids.
Current edition approved Oct. 1, 2018Oct. 1, 2023. Published October 2018October 2023. Originally approved in 2007. Last previous edition approved in 20132018 as
E2564 – 13.E2564 – 18. DOI: 10.1520/E2564-18.10.1520/E2564-23.
2
Gilchrist, Mary J. R., “Biosafety Precautions for Airborne Pathogens,” in Laboratory Safety: Principles and Practices, ASM Press, 1995, pp. 67–76.
3
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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E2564 − 23
3.2.3 microscopic factor (MF), n—a calibrated conversion factor for calculating the mycobacterium count per mL sample.
3.2.3.1 Discussion—
The average number of mycobacterium cells per one microscopic field (or oil field, OIF) is multiplied by the MF to give the
concentration of mycobacteria per mL of sample.
3.2.4 oil immersion field (OIF), n—the circular area of a microscopic field visible in the eyepiece of the microscope using oil
immersion objective.
4. Summary of Practice
4.1 This practice describes a semi-quantitative test for enumerating acid-fast stained environmental mycobacteria (AFB) from
4
metalworking fluids by direct microscopic counting (DMC) method. It is used to determine total mycobacterium counts, including
culturable and possibly dead or moribund cells in the sample. This practice cannot be used to determine the total viable
2
mycobacterium population in the sample. A known sample volume (centrifuged or direct) is spread over a known area (1 cm or
5
similar) on a microscope slide (marked by frosted or painted circles). Following differential acid-fast stai
...
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