ASTM E2564-13
(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 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. The 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.
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. For additional safety information, see Laboratory Safety: Principle and Practices, 4th Edition2
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Designation: E2564 − 13 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.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3.1.1.1 Discussion—Oncestained,mycobacteriumresistde-
colorization when exposed to acidified organic solvents, and
1.1 This practice describes a direct microscopic counting
are therefore, informally designated acid-fast.
method (DMC) for the enumeration of the acid fast stained
3.1.2 non-tuberculous Mycobacteria (NTM)—
mycobacteriapopulationinmetalworkingfluids.Itcanbeused
environmental mycobacteria, not associated with tuberculosis.
to detect levels of total mycobacteria population, including
culturable as well as non-culturable (possibly dead or mori-
3.1.3 microscopic factor (MF), n—a calibrated conversion
bund ) bacterial cells. This practice is recommended for all
factorforcalculatingtheMycobacteriumcountpermLsample.
water-based metalworking fluids.
3.1.3.1 Discussion—Theaveragenumberofmycobacterium
cells per one microscopic field (or oil field, OIF) is multiplied
1.2 This standard does not purport to address all of the
by the MF to give the concentration of mycobacterium per mL
safety concerns, if any, associated with its use. It is the
of sample.
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica-
3.1.4 oil immersion field (OIF), n—the circular area of a
bility of regulatory limitations prior to use. For additional
microscopic field visible in the eye piece of the microscope
safety information, see Laboratory Safety: Principle and
using oil immersion objective.
2
Practices, 4th Edition.
4. Summary of Practice
1.3 This international standard was developed in accor-
dance with internationally recognized principles on standard-
4.1 The practice describes a semi quantitative test for
ization established in the Decision on Principles for the
enumerating acid fast stained environmental mycobacterium
Development of International Standards, Guides and Recom-
(AFB) from metal working fluids by direct microscopic count-
4
mendations issued by the World Trade Organization Technical
ing (DMC) method. It is used to determine total mycobacte-
Barriers to Trade (TBT) Committee.
rium counts including culturable and possibly dead or mori-
bund cells in the sample. This practice cannot be used to
2. Referenced Documents
determine the total viable mycobacterium population in the
3
2.1 ASTM Standards:
sample. A known sample volume (centrifuged or direct) is
2
D2881Classification for Metalworking Fluids and Related
spread over a known area (1 cm or similar) on a microscope
Materials
slide (marked by frosted or painted circles). Following differ-
5
ential acid-fast staining, the acid-fast cells are counted in
3. Terminology
several microscopic fields over the designated area. The
3.1 Definitions of Terms Specific to This Standard: calculation is based on using a calibrated microscope with a
3.1.1 acid-fast bacteria, n—a distinctive staining property knownMicroscopicFactor(MF).TheMFisdeterminedbythe
of Mycobacteria due to their lipid-rich cell walls. 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
1
This practice is under the jurisdiction of ASTM Committee E34 on Occupa-
stained mycobacterium cells per microscopic field multiplied
tionalHealthandSafetyandisthedirectresponsibilityofSubcommitteeE34.50on
Health and Safety Standards for Metal Working Fluids.
bytheMFgivesthemycobacteriumnumberpermLofsample.
CurrenteditionapprovedJuly1,2013.PublishedJuly2013.Originallyapproved
in 2007. Last previous edition approved in 2011 as E2564-11. DOI: 10.1520/
E2564-13.
2 4
Mary J. R. Gilchrist, “Biosafety Precautions for Airborne Pathogens,” in Standard Methods for the Examination of Dairy Products, Chapter: 10: Direct
Laboratory Safety Principles and Practices, pp. 67–76, 1995, ASM Press. MicroscopicMethodsforBacteriaorSomaticCells,16thed.AmericaPublicHealth
3
For referenced ASTM standards, visit the ASTM website, www.astm.org, or Association, Inc., Washington, DC, 1978.
5
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Ebersole L. L., “Acid-Fast Stain Procedures,” pp. 3.5.1–3.5.11. In Clinical
Standards volume information, refer to the standard’s Document Summary page on Microbiology Procedures Handbook, Vol. 1. American Society for
...
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 − 11 E2564 − 13 An American National Standard
Standard Test Method 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
1.1 This test method 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 test method is recommended for all
water-based metalworking fluids.
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
2
limitations prior to use. For additional safety information, see Laboratory Safety: Principle and Practices, 4th Edition
2. Referenced Documents
3
2.1 ASTM Standards:
D2881 Classification for Metalworking Fluids and Related Materials
3. Terminology
3.1 Definitions of Terms Specific to This Standard:
3.1.1 acid-fast bacteria, n—a distinctive staining property of Mycobacteria due to their lipid-rich cell walls.
1
This test method 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 Jan. 1, 2011July 1, 2013. Published January 2011July 2013. Originally approved in 2007. Last previous edition approved in 20072011 as
E2564 - 07.E2564 - 11. DOI: 10.1520/E2564-11.10.1520/E2564-13.
2
Mary J. R. Gilchrist: Biosafety Precautions for Airborne Pathogens, in Laboratory Safety Principles and Practices, pp. 67-76, 1995, ASM Press
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.
3.1.1.1 Discussion—
Once stained, mycobacterium resist decolorization when exposed to acidified organic solvents, and are therefore, informally
designated acid-fast.
3.1.2 non-tuberculous Mycobacteria (NTM)—environmental mycobacteria, not associated with tuberculosis.
3.1.3 microscopic factor (MF), n—a calibrated conversion factor for calculating the Mycobacterium count per mL sample.
3.1.3.1 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 mycobacterium per mL of sample.
3.1.4 oil immersion field (OIF), n—the circular area of a microscopic field visible in the eye piece of the microscope using oil
immersion objective.
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E2564 − 13
4. Summary of Test Method
4.1 The method describes a semi quantitative test for enumerating acid fast stained environmental mycobacterium (AFB) from
4
metal working 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 test method 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 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
...
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