ASTM E2564-23

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Designation: E2564 − 18 E2564 − 23 An American National Standard
Standard Practice for
Enumeration of Mycobacteria in Metalworking Fluids by
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
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
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.
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.
Gilchrist, Mary J. R., “Biosafety Precautions for Airborne Pathogens,” in Laboratory Safety: Principles and Practices, ASM Press, 1995, pp. 67–76.
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
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
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
mycobacterium population in the sample. A known sample volume (centrifuged or direct) is spread over a known area (1 cm or
similar) on a microscope slide (marked by frosted or painted circles). Following differential acid-fast staining, the acid-fast cells
are counted in several microscopic fields over the designated area. The calculation is based on using a calibrated microscope with
a known microscopic factor (MF). The MF is determined by the microscopic area over which a known amount of sample was
spread, the number of microscopic fields in the marked circle, and the volume of sample examined. The number of acid-fast stained
mycobacterium cells per microscopic field multiplied by the MF gives the mycobacterium number per mL of sample.
5. Significance and Use
5.1 During the past decade, it has become increasingly apparent that non-tuberculous mycobacteria are common members of the
indigenous MWF bacterial population. 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.
6. Interferences
6.1 Some metalworking fluid formulations fail to completely dry or provide an uneven film on the microscope slide (for example,
synthetic fluids and metalworking fluids with high trap tramp oil content and debris). For these samples, the results can be difficult
to interpret, as heat fixing may not provide full adherence. These samples should be re-stained or a new slide may be prepared.
6.2 A negative acid-fast staining reaction does not necessarily indicate that a sample will be culturally negative for Mycobacteria,
since the culture method has a lower detection limit (1 cell/mL) than the DMC method.
Standard Methods for the Examination of Dairy Products, Chapter: 10: Direct Microscopic Methods for Bacteria or Somatic Cells, 16th ed., American Public Health
Association, Inc., Washington, DC, 1978.
Ebersole, L. L., “Acid-Fast Stain Procedures,” pp. 3.5.1–3.5.11, in Clinical Microbiology Procedures Handbook, Vol 1, American Society for Microbiology, Washington,
DC, 1994.
E2564 − 23
7. Apparatus
7.1 Centrifuge, (“microfuge”), 14 000 relative gravities.
7.2 Centrifuge Tubes with Caps, disposable, 1 mL to 2 mL capacity, such as Eppendorf Safe-Lock tube or any other suitable
centrifuge tubes.
7.3 Calibrated Variable Pipet, with sterile tips: 5 μL, 10 μL, 1.0 mL, 5 mL.
7.4 Microscope Slides, with 100 mm or similar areas, marked by frosted or painted circles and frosted labeling ends.
7.5 Calibrated Stage Micrometer, 0.01 mm or similar divisions.
7.6 Compound Micro
...